Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.Despite the importance of cell surface biology, the conventional shotgun proteomics strategy generally underrepresents the membrane proteome because of inadequate solubilization and protease digestion (1, 2). The ageless gel strategy, consisting of SDS-PAGE followed by in-gel digestion, can partially solve this problem (3–5), but the recovery from in-gel digestion is generally lower than that from in-solution digestion, and this approach is far from suitable for a rapid, simple, and high throughput automated system. Numerous approaches have been reported to overcome the difficulties in membrane proteome analysis, such as the use of surfactants (2, 6–11), organic solvents (6, 7, 12–15), or chaotropic reagents (2, 6, 16). Acid-labile surfactants, such as RapiGest SF, are among the most promising additives to enhance protein solubilization without interfering with LC-MS performance (6, 10, 17–19). However, the cleavage step at acidic pH causes loss of hydrophobic peptides because of coprecipitation with the hydrophobic part of RapiGest SF (20). Recently, we developed a new protocol to dissolve and digest membrane proteins with the aid of a removable phase transfer surfactant (PTS),1 such as sodium deoxycholate (SDC) (20). The solubility of membrane proteins with SDC was comparable to that with sodium dodecyl sulfate. In addition, the activity of trypsin was enhanced ∼5-fold in the presence of 1% SDC because this rapid PTS method mimics conditions in the alimentary canal in which bile salts such as cholate and deoxycholate are secreted together with trypsin. After tryptic digestion, SDC is removed prior to LC-MS/MS analysis by adding an organic solvent followed by pH-induced transfer of the surfactant to the organic phase, whereas tryptic peptides remain in the aqueous phase. This protocol offers a significant improvement in identifying membrane proteins by increasing the recovery of hydrophobic tryptic peptides compared with the protocols using urea and RapiGest SF.The goal of this study is to establish a membrane proteomics method that is unbiased with respect to protein solubility, hydrophobicity, and protein abundance; i.e. membrane proteins can be as efficiently extracted and digested as soluble proteins. So far, to our knowledge, little information about the recovery of the membrane proteome has been reported. Instead, the number of identified membrane proteins or the content of membrane proteins identified in the membrane-enriched fraction has been used as an indicator of the efficiency of procedures for membrane proteome analysis (4, 5, 21–23). However, these parameters usually depend on the experimental conditions, including the sample preparation procedure and LC-MS instrument used. Therefore, it is difficult to compare data obtained with these protocols except in the case of direct comparison. Furthermore, there has been no report quantitatively comparing the recovery of membrane proteome with that of soluble proteins.In this study, we used a modified version of our PTS protocol with immobilized trypsin columns to reduce the digestion time and evaluated its suitability for unbiased quantitation of the membrane proteome. In addition, we applied this protocol to estimate the copy numbers per cell of 1,453 proteins, including 545 membrane proteins, using the exponentially modified protein abundance index (emPAI). Finally, this rapid and unbiased PTS protocol was applied to the quantitative analysis of Escherichia coli BW25113 wild-type and relA knock-out (KO) strains.     

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD)¹ is severe synaptic loss (1–3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10–12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13–18). Post-translational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC–tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and age-matched controls, as well as a study of SNARE complex protein levels in brain tissue from prion-infected mice.     

Intermembrane Space Proteome of Yeast Mitochondria

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.Mitochondria are double-membrane-bound organelles that fulfill a multitude of important cellular functions. Proteomic analysis of purified mitochondria revealed that they contain approximately 1000 (yeast) to 1500 (human) different proteins (1–3). However, the distribution of these proteins among the four mitochondrial subcompartments (outer membrane, inner membrane, matrix, and intermembrane space) has been only marginally studied through global approaches. This is attributed to the high complexity of purifying submitochondrial fractions to a grade suitable for proteomic analysis. The best-studied submitochondrial proteomes comprise the outer membranes of S. cerevisae, N. crassa, and A. thaliana (4–6). The mitochondrial intermembrane space (IMS)¹ represents a highly interesting compartment for several reasons: it provides a redox active space that promotes oxidation of cysteine residues similar to the endoplasmic reticulum and the bacterial periplasm, but unlike cytosol, nucleus, or the mitochondrial matrix where the presence of thioredoxins or glutaredoxins prevents the risk of unwanted cysteine oxidation (7, 8). Furthermore in higher eukaryotes IMS proteins are released into the cytosol upon apoptotic induction, which triggers the activation of a cell-killing protease activation cascade (9, 10). The IMS can also exchange proteins, lipids, metal ions, and various metabolites with other cellular compartments, allowing mitochondrial metabolism to adapt to cellular homeostasis. In particular, the biogenesis and activity of the respiratory chain were shown to be controlled by various proteins of the IMS (11–13). Most of the currently known IMS proteins are soluble proteins; however, some inner membrane proteins have been annotated as IMS proteins as well, such as proteins that are peripherally attached to the inner membrane or membrane proteins that expose enzyme activity toward the IMS (8).All IMS proteins are encoded in the nuclear DNA and have to be imported after translation in the cytosol (14–19). Two main pathways are known to mediate the import and sorting of proteins into the IMS. One class of proteins contains bipartite presequences that consist of a matrix targeting signal and a hydrophobic sorting signal. These signals arrest the incoming preprotein at the inner membrane translocase TIM23. After insertion into the inner membrane, the soluble, mature protein can be released into the IMS by the inner membrane protease (IMP) (20–22). The second class of IMS proteins possesses characteristic cysteine motifs that typically are either twin CX₉C or twin CX₃C motifs (23, 24). Upon translocation across the outer membrane via the TOM complex, disulfide bonds are formed within the preproteins, which traps them in the IMS. Disulfide bond formation is mediated by the MIA machinery, which consists of the inner-membrane-anchored Mia40 and the soluble IMS protein Erv1 (25–28).The release of cytochrome c from the IMS upon binding and insertion of Bax at the outer membrane is a hallmark of programmed cell death. Although Bax is found only in higher eukaryotes, it was shown that recombinant mammalian Bax induces the release of cytochrome c upon incubation with isolated yeast mitochondria (29, 30). Furthermore, we found that not only cytochrome c but also other soluble IMS proteins are released from Bax-treated yeast mitochondria, whereas soluble matrix proteins largely remain within the organelle (30).We used this apparently conserved mechanism to systematically profile the protein composition of the yeast mitochondrial IMS by employing an experimental approach based on stable isotope labeling, which allowed for the specific identification of Bax-dependent protein release. Almost the entire set of known soluble IMS proteins was identified, and 20 additional, novel soluble IMS proteins were found. We confirmed IMS localization for 15 proteins through biochemical assays. Among these proteins, we identified novel proteins that fall into several classes: (i) those that are involved in maintaining protein redox homeostasis (thioredoxins, thioredoxin reductases, or thiol peroxidases), (ii) those that undergo proteolytic processing by IMP (Ptc5), (iii) those that utilize the MIA pathway for their import (Mic23 and Coa6), and (iv) those that play a role in the assembly of cytochrome c oxidase (Coa6).     

Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction,Molecular Transport,and Callose Biosynthesis

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.The plasma membrane (PM)¹ is considered as one of the most interactive and dynamic supramolecular structures of the cell (1, 2). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4–8). Typically, PM proteins are either embedded in the phospholipid bilayer through transmembrane helices or less tightly bound to the membrane through reversible or irreversible surface interactions. In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9, 10). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9, 10). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9–11). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10, 11). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM.Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13–15), Arabidopsis (16), barrel clover (Medicago truncatula) (17), rice (18), oat, and rye (19). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15–19). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15–19). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells.Although several reports describe the partial characterization of DRMs from higher plants (13–23), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones.Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1, 2). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its “effectors” (3), and SNARE³ (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5), termed “cis-SNARE complexes,” if they are all anchored to the same membrane bilayer, or “trans-SNARE complexes” if they are anchored to apposed membranes.Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6), high levels of calcium (7), viral-encoded fusion proteins (8, 9), or SNAREs (10, 11). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12–15), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion.We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17), and key “regulatory” lipids including ERG, phosphoinositides, and DAG (18). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17, 20–22) and proofreads correct vacuolar SNARE pairing (23). HOPS also has direct affinity for phosphoinositides (17). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24). Docking requires Ypt7p (25) and HOPS (17). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26) and lipids (18) required for fusion becomes enriched in a ring-shaped microdomain, the “vertex ring,” which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18, 27). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28), in accord with model liposome studies (14). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies.Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29), this was not stimulated by HOPS and Sec17p/Sec18p (30). SNARE proteoliposomes bearing all the vacuolar lipids (18, 31–33), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli.The cells of living organisms contain different types of membranes performing uniquely specific functions that are largely dictated by their protein compositions. Membrane proteome typically contains integral membrane proteins (IMPs)¹ with one or more transmembrane domains (TM) and peripheral membrane proteins (PMPs) without TM. PMPs usually interact with IMPs and function together as protein complexes, as typically demonstrated by the peripheral subunits of the membrane protein complexes such as photosystem (PS) I, PSII, the F₁F₀-ATP synthase, and ABC type transporters (1–6). Identification of the PMPs is important for the understanding of the underlying mechanism of various membrane related functions, and could help to discover novel functionally important membrane protein complexes.Large-scale identification of PMPs were typically performed by identification of the total proteins from the isolated whole membranes from which PMPs were predicted by the absence of TM using topology prediction software such as TMHMM (7), or by identification of the proteins extracted from the intact whole membranes with chaotropic reagents such as high concentration salts, urea, or high pH solution (8–13). These methods can identify some non-TM containing proteins uniquely from the membrane fraction. However, in most cases the majority of the non-TM containing proteins identified with such methods can also be identified from the soluble fraction that is expected to consist of mainly cytoplasmic proteins. Therefore, it is necessary to evaluate whether the non-TM containing proteins identified from the membranes are true PMPs or just some carry-over contaminant from the soluble fraction during sample fractionation. Unfortunately, the high throughput method to perform such an evaluation is still lacking, and such a method is a pressing need considering the ever-increasing number of identified proteins from a single proteomic study.The unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) is an ideal organism for studies in membrane proteomics. Synechocystis is the first cyanobacterium with a completely sequenced genome and contains large numbers of membrane structures (12–14). The organism can naturally take up foreign DNA from environment and integrate it into its genome through homologous recombination, making it simple to perform target mutagenesis for the validation of functional significance of proteins screened from high throughput approaches. The autotrophic growth ability allows Synechocystis to emerge as a potential cost-effective cell factory for producing clean and renewable biofuels to deal with the world-wide crisis of energy shortage and environmental pollution (15–18). Functional proteomics have great potential in the identification of novel target proteins and for discovering and optimizing novel protein networks for the generation of biofuel-producing strains with higher efficiency and less cost.We separated Synechocystis whole cell lysates into membrane and soluble fractions, and identified the proteins in each fraction with unprecedented coverage using high-resolution MS. We present a novel method and its rationale for evaluating the tightness of membrane association for all non-TM containing proteins identified in both fractions. This built a foundation for the large-scale identification of bona fide peripheral membrane proteins, particularly for the hypothetical and unknown proteins that are not known to be physically or functionally associated with the membranes.     

The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome: IDENTIFICATION, GLYCOSITE OCCUPANCY, AND MEMBRANE ORIENTATION*

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function. One of the best examples of endogenous repair mechanisms involves skeletal muscle, which has innate regenerative capacity (for reviews, see Refs. 1–4). Skeletal muscle repair begins with satellite cells, a heterogeneous population of mitotically quiescent cells located in the basal lamina that surrounds adult skeletal myofibers (5, 6), that, when activated, rapidly proliferate (7). The progeny of activated satellite cells, known as myogenic precursor cells or myoblasts, undergo several rounds of division prior to withdrawal from the cell cycle. This is followed by fusion to form terminally differentiated multinucleated myotubes and skeletal myofibers (7, 8). These cells effectively repair or replace damaged cells or contribute to an increase in skeletal muscle mass.The molecular mechanisms that regulate differentiation of satellite cells and myoblasts toward myofibers are not fully understood, although it is known that the cell surface proteome plays an important biological role in skeletal muscle differentiation. Examples include how cell surface proteins modulate myoblast elongation, orientation, and fusion (for a review, see Ref. 8). The organization and fusion of myoblasts is mediated, in part, by cadherins (for reviews, see Refs. 9 and 10), which enhance skeletal muscle differentiation and are implicated in myoblast fusion (11). Neogenin, another cell surface protein, is also a likely regulator of myotube formation via the netrin ligand signal transduction pathway (12, 13), and the family of sphingosine 1-phosphate receptors (Edg receptors) are known key signal transduction molecules involved in regulating myogenic differentiation (14–17). Given the important role of these proteins, identifying and characterizing the cell surface proteins present on myoblasts in a more comprehensive approach could provide insights into the molecular mechanisms involved in skeletal muscle development and repair. The identification of naturally occurring cell surface proteins (i.e. markers) could also foster the enrichment and/or characterization of cell intermediates during differentiation that could be useful therapeutically.Although it is possible to use techniques such as flow cytometry, antibody arrays, and microscopy to probe for known proteins on the cell surface in discrete populations, these methods rely on a priori knowledge of the proteins present on the cell surface and the availability/specificity of an antibody. Proteomics approaches coupled with mass spectrometry offer an alternative approach that is antibody-independent and allows for the de novo discovery of proteins on the surface. One approach, which was used in the current study, exploits the fact that a majority of the cell surface proteins are glycosylated (18). The method uses hydrazide chemistry (19) to immobilize and enrich for glycoproteins/glycopeptides, and previous studies using this chemistry have successfully identified soluble glycoproteins (20–24) as well as cell surface glycoproteins (25–28). A recently optimized hydrazide chemistry strategy by Wollscheid et al. (29) termed cell surface capturing (CSC)¹ technology, reports the ability to identify cell surface (plasma membrane) proteins specifically with little (<15%) contamination from non-cell surface proteins. The specificity stems from the fact that the oligosaccharide structure is labeled using membrane-impermeable reagents while the cells are intact rather than after cell lysis. Consequently, only extracellular oligosaccharides are labeled and subsequently captured. Utilizing information regarding the glycosylation site then allows for a rapid elimination of nonspecifically captured proteins (i.e. non-cell surface proteins) during the data analysis process, a feature that makes this approach unique to methods where no label or tag is used. Additionally, the CSC technology provides information about glycosylation site occupancy (i.e. whether a potential N-linked glycosylation site is actually glycosylated), which is important for determining the protein orientation within the membrane and, therefore, antigen selection and antibody design.To uncover information about the cell surface of myoblasts and to identify potential markers of myoblast differentiation, we used the CSC technology on the mouse myoblast C2C12 cell line model system (30, 31). This adherent cell line derived from satellite cells has routinely been used as a model for skeletal muscle development (e.g. Refs. 1, 32, and 33), skeletal muscle differentiation (e.g. Refs. 34–36), and studying muscular dystrophy (e.g. Refs. 37–39). Additionally, these cells have been used in cell-based therapies (e.g. Refs. 40–42). Using the CSC technology, 128 cell surface N-linked glycoproteins were identified, including several that were found to change in overall abundance as the myoblasts differentiate toward myotubes. The current data also confirmed the occupancy of 235 N-linked glycosites of which 226 were previously unconfirmed. The new information provided by the current study is expected to facilitate the development of useful tools for studying the differentiation of myoblasts toward myotubes.     

Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes

The human stomatin-like protein-1 (SLP-1) is a membrane protein with a characteristic bipartite structure containing a stomatin domain and a sterol carrier protein-2 (SCP-2) domain. This structure suggests a role for SLP-1 in sterol/lipid transfer and transport. Because SLP-1 has not been investigated, we first studied the molecular and cell biological characteristics of the expressed protein. We show here that SLP-1 localizes to the late endosomal compartment, like stomatin. Unlike stomatin, SLP-1 does not localize to the plasma membrane. Overexpression of SLP-1 leads to the redistribution of stomatin from the plasma membrane to late endosomes suggesting a complex formation between these proteins. We found that the targeting of SLP-1 to late endosomes is caused by a GYXXΦ (Φ being a bulky, hydrophobic amino acid) sorting signal at the N terminus. Mutation of this signal results in plasma membrane localization. SLP-1 and stomatin co-localize in the late endosomal compartment, they co-immunoprecipitate, thus showing a direct interaction, and they associate with detergent-resistant membranes. In accordance with the proposed lipid transfer function, we show that, under conditions of blocked cholesterol efflux from late endosomes, SLP-1 induces the formation of enlarged, cholesterol-filled, weakly LAMP-2-positive, acidic vesicles in the perinuclear region. This massive cholesterol accumulation clearly depends on the SCP-2 domain of SLP-1, suggesting a role for this domain in cholesterol transfer to late endosomes.Human stomatin-like protein-1 (SLP-1),³ also known as STOML-1, STORP (1), slipin-1 (2), or hUNC-24 (3), is the human orthologue of Caenorhabditis elegans UNC-24 and a member of the stomatin protein family that comprises 5 human members: stomatin (4–6), SLP-1 (1, 7), SLP-2 (8), SLP-3 (9, 10), and podocin (11). SLP-1 is predominantly expressed in the brain, heart, and skeletal muscle (7, 8) and can be identified in most other tissues (1). Its structure contains a hydrophilic N terminus, a 30-residue hydrophobic domain that is thought to anchor the protein to the cytoplasmic side of the membrane, followed by a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (12) that is also known as prohibitin (PHB) domain (13), and a C-terminal sterol carrier protein-2 (SCP-2)/nonspecific lipid transfer protein domain (14, 15). This unique structure that was first revealed in C. elegans UNC-24 (16) suggests that SLP-1 may be involved in lipid transfer and transport (17).The founder of the family, stomatin, is a major protein of the red blood cell membrane (band 7.2) and is ubiquitously expressed (18). It is missing in red cells of patients with overhydrated hereditary stomatocytosis, a pathological condition characterized by increased permeability of the red cells for monovalent ions and stomatocytic morphology (19, 20). However, the lack of stomatin is not due to a mutation in its gene but rather to a transport defect (21, 22). Stomatin is a monotopic, oligomeric, palmitoylated, cholesterol-binding membrane protein (18) that is associated with lipid rafts (23, 24) or raft-like detergent-resistant membranes (DRMs) (25), serving as a respective marker (26–28). Other stomatin family members like podocin (29, 30) and SLP-3 (9) are also enriched in DRMs. Many SPFH/PHB proteins share this property suggesting that the SPFH/PHB domain plays an important role in lipid raft/DRM targeting (13, 31). Several interactions of stomatin with membrane proteins have been revealed, notably with the acid sensing ion channels (32) and the glucose transporter GLUT1 (33, 34). Interestingly, stomatin functions as a switch of GLUT1 specificity from glucose to dehydroascorbate in the human red blood cell thus increasing vitamin C recycling and compensating the human inability to synthesize vitamin C (35).The C. elegans genome contains 10 members of the stomatin family. Defects in three of these genes (mec-2, unc-1, and unc-24) cause distinct neuropathologic phenotypes, namely uncoordinated movement and defect in mechanosensation, respectively (36, 37). These are explained by dysfunction of the respective stomatin-like proteins in complex with degenerin/epithelial sodium channels that also affects the sensitivity to volatile anesthetics (38, 39). Importantly, MEC-2 and human podocin bind cholesterol and form large supercomplexes with various ion channels thus modulating channel activity (40). The biological functions of the SLP-1 orthologue UNC-24 and stomatin orthologue UNC-1 are associated, because the unc-24 gene controls the distribution or stability of the UNC-1 protein (41). In addition, UNC-24 co-localizes and interacts with MEC-2 and is essential for touch sensitivity (36). Based on these observations, we hypothesize that human stomatin and SLP-1 similarly interact and modify the distribution of each other. These proteins may have important functions in regulating the activity of ion channels in the human brain and muscle tissues. Despite its putative role in cellular lipid distribution, SLP-1 has not been studied to date.In this work, we characterized human SLP-1 as a late endosomal protein and identified an N-terminal GYXXΦ motif as the targeting signal. We found that SLP-1 interacts with stomatin in vitro and in vivo and associates with DRMs. Regarding the proposed lipid transfer function, we showed that SLP-1 induces the formation of large, cholesterol-rich vesicles or vacuoles when cholesterol trafficking from the late endosomes is blocked suggesting a net cholesterol transfer to the late endosomes and/or lysosomes. This effect was clearly attributed to the SCP-2/nonspecific lipid transfer protein domain of SLP-1, in line with the original hypothesis.     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcA/ΔmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s⁻¹). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.Respiratory electron flow typically occurs at the inner membrane, where oxidation and reduction can be easily linked to intracellular electron carriers and used to generate a membrane potential. However, when the electron acceptor or donor is insoluble, bacteria must possess a mechanism for transferring electrons beyond their inner membrane (1). This is especially true for Proteobcteria, which have an outer membrane that further insulates cytoplasmic and inner membrane processes from insoluble substrates. Metal oxides (such as Fe(III) and Mn(IV) oxyhydroxides) are well recognized naturally occurring electron acceptors that demand such an electron transfer strategy (2–4).Shewanella oneidensis MR-1, a metabolically versatile member of the gammaproteobacteria (5), is capable of reducing insoluble metals, and this phenotype has been linked to a collection of interacting multiheme cytochromes spanning the inner membrane, periplasmic space, and outer membrane (6–12). There is also evidence that some of these cytochromes decorate the surface of pili-like structures extending from the cell surface (13, 14). Regardless of the ultimate location of the cytochromes, in all models of electron transfer, electrons must hop from these proteins to a solid surface or be transferred to a soluble mediator that can diffuse to a final destination (15, 16). Although chelation of a metal oxide is a third option (17, 18), the fact that Shewanella is able to transfer electrons to solid graphite electrodes (19–23) underscores the need for a direct or diffusion-based electron transfer mechanism to link cellular proteins and surfaces.Recent work has shown that Shewanella species secrete soluble flavins (FMN and riboflavin) that facilitate electron transfer to both metals and electrodes (23, 24). For example, removal of accumulated soluble flavins decreases the rate of electron transfer by Shewanella biofilms to electrodes over 80%. Consistent with this observation, kinetic measurements with pure MtrC and OmcA (25) showed that direct reduction of solid metal oxides by these cytochromes was too slow to explain physiological rates of electron transfer, whereas turnover rates of these enzymes with soluble flavins were orders of magnitude larger. These studies suggest that the kinetics of electron transfer from cytochromes on the outer surface of Shewanella to electrodes will be significantly altered in the absence of diffusible mediators (9–11, 26–34).Voltammetry has proven a useful technique for the analysis of electron transfer rates and pathways using purified proteins (35–39) and has recently been extended to the study of intact bacteria (23, 40–42). In slow scan rate cyclic voltammetry, the rate of electron transfer from respiring Shewanella biofilms to electrodes rises sharply at the E°′ of riboflavin and FMN (−0.2 V versus SHE)² (23). Such measurements relating thermodynamic driving force to turnover kinetics would be difficult with whole cell:Fe(III) oxide incubations, which do not allow fine control over the electron acceptor redox potential or real time recording of electron transfer rates. In addition, voltammetry provides tools to observe electron movement under single-turnover conditions (in the absence of electron donor), allowing reversible oxidation and reduction of proteins accessible to the electrode and study of kinetic behavior (43, 44).In this work, techniques of turnover (sustained electron transfer from cells to electrode in the presence of electron donor) and single turnover (reversible oxidation and reduction in the absence of electron donor) voltammetry were harnessed to investigate the role of outer membrane proteins in electron transfer from Shewanella to electrodes. In all of these studies, intact metabolically active cells were used, along with electrode surfaces known to act as acceptors for Shewanella. The results in the absence of soluble mediators provide evidence that electron transfer between MtrC and OmcA and surfaces requires a higher potential compared with when flavins are present to shuttle electrons to the surface. Mutant analysis also demonstrates that cells possessing different outer membrane cytochromes have differing abilities for direct and mediator-enabled electron transfer.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets

Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC physiology, which may represent promising targets for designing new therapeutic interventions.Neisseria gonorrhoeae (GC)¹ is an obligate human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. GC infection remains a significant health and economic burden worldwide (1). It is also the second most commonly reported infectious disease in the United States (2). Gonorrhea ranges from clinically asymptomatic to local genital infections to disseminated bloodstream infections. Asymptomatic infections often have devastating consequences on women''s health including pelvic inflammatory disease, ectopic pregnancy, and infertility (3). Additionally, GC infections facilitate transmission and acquisition of HIV (4). For all of these reasons it is critical to provide effective treatments against gonorrhea. Currently, a dual therapy with cephalosporin and either azithromycin or doxycycline is recommended (5). However, over the past several years treatment failures associated with GC strains displaying decreased susceptibility to extended spectrum cephalosporins have been reported from various parts of the world (6–9). This is especially concerning because no other antibiotics are clinically useful in these cases, and because no appropriate vaccine exists (10). The escalating problem of the spread of antimicrobial resistance in GC, and the importance of the development of new approaches to study, treat, and prevent GC infection, have been recognized by the World Health Organization and by the Centers for Disease Control and Prevention (11, 12).We propose that largely unexplored proteins localized to bacterial cell envelope and naturally released membrane vesicles are particularly promising as potential novel molecular targets for therapeutic interventions against gonorrhea. The small fraction of known components of the GC cell envelope (outer membrane, periplasm, cytoplasmic membrane) plays a fundamental role in establishing infection by enabling the microbes to adhere to and invade host cells, facilitating nutrient acquisition, host tissue destruction, and suppression of immune responses (3, 13–15). Further, GC, like many other Gram-negative bacteria, produces membrane vesicles (MVs), which are nano-sized bilayered proteolipids (16). Naturally produced MVs are potentially an effective way to deliver toxins, enzymes, and other effectors to host tissues. Additionally, evidence from various studies support that MVs participate in intercellular communication and horizontal gene transfer (16–21). In GC, MVs are necessary for biofilm formation, which is thought to play an important role in asymptomatic infection in women, resistance to antimicrobial agents, and suppression of host immune defenses (22–24). MVs may also contribute to the serum resistance by providing an enhanced ability to bind and eliminate bactericidal factors (17).Although the potential importance of proteins localized to the GC cell envelope and MVs has been reported previously (25, 26), only two proteomic studies have been published addressing GC membrane composition (27, 28). Most studies have focused on extensive characterization of factors involved in direct host cell interaction: protruding surface proteins (pili), outer membrane adhesins Opa, porins P.IA and P.IB, lipooligosaccharide, and several iron utilization proteins (3, 4, 15, 29–32). Many of these vital virulence factors undergo phase and/or antigenic variation, making them poor drug or vaccine targets. Therefore, the pursuit for novel and constitutively expressed proteins—therapeutic targets in GC—is of utmost importance.Accordingly, in this study we applied global and unbiased proteomics to compare the composition of both the cell envelopes and MVs isolated from four GC strains: FA1090, F62, MS11, and 1291. Specifically, we used isotope tagging for relative and absolute quantification (iTRAQ) coupled with multidimensional liquid chromatography and tandem mass spectrometry (2D-LC/MS/MS). This approach allowed us to determine a uniformly and differentially expressed repertoire of proteins. Focusing on a homolog of LPS transport protein, LptD (OstA, Imp), which was identified in both the cell envelopes and MVs fractions, and ubiquitously expressed among analyzed strains, we showed that its depletion led to loss of GC viability. Finally, we selected for initial characterization six predicted outer membrane proteins, which were present at similar levels in the GC cell envelopes. We generated Δngo1344, Δngo1955, Δngo1985, Δngo2111, Δngo2121, and Δngo2139 mutant strains and examined their sensitivity toward different cell envelope-perturbing agents as well as chloramphenicol. These studies showed that the lack of NGO1985 resulted in dramatically decreased GC viability, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Overall, these findings further support our hypothesis that the conserved proteins may represent promising targets for designing new therapeutic interventions.