F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry

Ribosomal protein S1 has been shown to be a significant effector of prokaryotic translation. The protein is in fact capable of efficiently initiating translation, regardless of the presence of a Shine-Dalgarno sequence in mRNA. Structural insights into this process have remained elusive, as S1 is recalcitrant to traditional techniques of structural analysis, such as x-ray crystallography. Through the application of protein cross-linking and high resolution mass spectrometry, we have detailed the ribosomal binding site of S1 and have observed evidence of its dynamics. Our results support a previous hypothesis that S1 acts as the mRNA catching arm of the prokaryotic ribosome. We also demonstrate that in solution the major domains of the 30S subunit are remarkably flexible, capable of moving 30–50Å with respect to one another.Initiation of translation is often the rate-limiting step of protein biosynthesis (1). In prokaryotes, this process is widely recognized to be directed by the Shine-Dalgarno (S.D.)¹ sequence of mRNA and its complementation with the 3′ end of 16S rRNA (2). However, binding of the S.D. sequence to the ribosome is not obligatory for initiation. Ribosomal protein S1, widely conserved in prokaryotes, (3) has been shown to efficiently initiate translation, regardless of the presence of an S.D. sequence (4, 5).S1 is a strikingly atyptical ribosomal protein, being both the largest (61 kDa) and the most acidic (pI 4.7) (6). The protein is composed of six homologous repeats each forming beta barrel domains (3) that in solution comprise a highly elongated structure spanning up to ca. 230 Å (7). This length is comparable to the diameter of the ribosome itself. In addition to these anomalous characteristics, S1 is also one of only two ribosomal proteins that has been attributed functional significance (6). Ribosomal protein S1, for instance, has no apparent role in the assembly of the ribosome, (2) yet is critical for translation in E. coli (8, 9). The functional significance of S1 is related to its most pronounced characteristic, the ability to simultaneously bind mRNA and the ribosome. Analysis of fragments produced by limited proteolysis and chemical cleavage of S1 has shown that an N-terminal fragment of S1 (residues 1–193) binds the ribosome (10) but not RNA (11). Likewise, a C-terminal fragment (res 172–557) binds RNA (12, 13) but not the ribosome (6, 10). By nature of this bi-functional structure, S1 enhances the E. coli ribosome''s affinity for RNA ∼5000 fold (14) and can directly mediate initiation of translation by binding the 5′ UTR of mRNA (4, 5). These observations have led to the hypothesis that S1 acts as a catching arm for the prokaryotic ribosome, working to bring mRNA to the proximity of the ribosome and thereby facilitate initiation (6).Unfortunately, structural analyses capturing how S1 is able to function in this manner remain elusive. A high-resolution crystal structure of ribosome bound S1, or even free S1, does not exist, because S1 is recalcitrant to crystallography (6). Preparation of ribosomes for x-ray crystallography actually involves the deliberate removal of ribosomal protein S1 as a means to improve the reproducibility of crystallization and the quality of the ribosome crystals formed (15–17). The structure and interactions of the protein have nevertheless intrigued structural biologists for decades. However, studies completed to date have failed to convincingly demonstrate the interaction between S1 and the rest of the 30S subunit, because they were incapable of localizing the individual S1 domains (16, 18–20).We have studied the binding of S1 to the 30S subunit by combining cross-linking with mass spectrometry. Chemical cross-linking has long been appreciated as a technique to probe protein-protein interactions (21, 22). With the advent of modern mass spectrometers, it can be very effectively employed to confidently identify the exact residues involved in linkages (23–28). In most cross-linking analyses, protein residues are targeted for covalent modification with a molecule that contains two reactive groups separated by a spacer arm of known length. Only protein residues closer than the length of the spacer arm are capable of being linked. Identification of cross-linked residues thereby provides distance constraints for structural modeling. In this work, the novel amidinating protein cross-linker, DEST (diethyl suberthioimidate), was employed (29, 30). This amine reactive reagent, unlike commercially available reagents, preserves the native basicity of the residues it modifies while being effective at physiological pH. Use of the reagent is unlikely to perturb protein structure and the modifications it imparts are compatible with ionization for mass spectrometry. We have additionally shown that the cross-links it forms can be efficiently enriched from other components of proteolytic digests using strong cation exchange (SCX) chromatography, (30) and that DEST cross-linking of ribosomes yields structural information in excellent agreement with x-ray crystallography (29). Although DEST is an 11Å spacer arm cross-linker, it links alpha carbons up to 24Å apart because of the length and flexibility of lysine side chains. Nevertheless, this is sufficient resolution to approximate the binding positions of the 10kDa domains of S1. Furthermore, multiple cross-linking of a single domain significantly enhances the resolution with which it can be localized.Here, through the application of protein cross-linking and high resolution mass spectrometry, we show that S1 binds to the 30S subunit near the anti-S.D. motif of the 16S rRNA, demonstrate that it is highly elongated even when bound to the ribosome, and provide evidence that its C-terminal mRNA binding region is remarkably dynamic. Our results thus indicate S1 is structurally poised, as previously hypothesized, (6) to act as the mRNA catching arm of the prokaryotic ribosome.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

LANCL2 Is Necessary for Abscisic Acid Binding and Signaling in Human Granulocytes and in Rat Insulinoma Cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38⁺/LANCL2⁺ HeLa transfected with a chimeric G-protein (Gα_q/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G_i. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.The plant hormone abscisic acid (ABA)⁴ plays a fundamental role in the regulation of plant response to environmental conditions, as well as in plant tissue development (1). Although the ABA biosynthetic pathway in plants and in fungi has been largely detailed, identification of the components of the ABA signaling pathway, particularly of the ABA receptor(s), has remained elusive. Two ABA-binding proteins have been identified in different plant tissues: the chloroplast Mg-chelatase subunit H (2) and, most recently, the G-protein-coupled receptor GCR2, which appears to mediate ABA-controlled stomatal closure and seed dormancy in Arabidopsis (3), although the role of GCR2 in the control of seed germination is still controversial (4–6) and its coupling to a G-protein has been refuted on the basis of sequence analyses (7–8). The Mg-chelatase subunit H was proposed as an intracellular ABA receptor, whereas GCR2 is a plasmamembrane protein, which interacts with the only Gα subunit (GPA 1) present in Arabidopsis (3). Although the Mg-chelatase subunit H does not show any significant homology with mammalian proteins, GCR2 shares a high amino acid identity with the mammalian peptide-modifying lanthionine synthetase C-like protein (LANCL) family (7). The animal LANCL protein family in turn shows structural similarities with the prokaryotic lanthionine synthetase component C proteins (9) involved in the synthesis of lanthionine-containing antimicrobial peptides known as lantibiotics (10).The fact that lantibiotics are not produced in animals suggests that LANCL proteins have a different function than prokaryotic lanthionine synthetase component C proteins. The human genome contains three LANCL genes, LANCL1, LANCL2, and LANCL3, located on chromosomes 2 and 7 and the X chromosome, respectively (11, 12). LANCL1 was the first member of the family to be isolated from human erythrocyte membranes (13). The LANCL2 mRNA was identified in a screening procedure for genes whose down-regulation resulted in anticancer drug resistance; thus, LANCL2 was also called testis-specific Adriamicin sensitivity protein (14). The structural assignment for the human LANCL proteins remains controversial. Based on the presence of seven putative transmembrane domains, LANCL1 and -2 were originally described as new G-protein-coupled receptors (GPCR69A and GPR69B, respectively); however, subsequent studies performed on human epithelial cells overexpressing LANCL1 or LANCL2 fused to the green fluorescent protein (LANCL1-GFP and LANCL2-GFP) showed that LANCL1-GFP is mainly found in the cytosol and in the nucleus, whereas LANCL2-GFP is associated with the plasmamembrane through N-terminal myristoylation (15). Similarly, the debate over the structurally related GCR2 is still open (3–6, 8).ABA has recently been demonstrated to be an endogenous pro-inflammatory hormone in human granulocytes, stimulating several cell functions (phagocytosis, reactive oxygen species and nitric oxide production, chemotaxis, and chemokinesis) through a pathway involving a pertussis toxin (PTX)-sensitive G-protein/receptor complex located on the plasmamembrane, cAMP overproduction, protein kinase A-dependent phosphorylation of the human ADP-ribosyl cyclase CD38, and consequent cADP-ribose (cADPR) generation, leading to an increase of the intracellular Ca²⁺ concentration (16; see also Ref. 17). This signaling pathway is similar to that triggered by ABA in plants (18). Fluorescence microscopy confirmed binding of biotinylated ABA to the granulocyte plasmamembrane. Scatchard plot analysis of [³H]ABA binding demonstrated presence of both high and low affinity ABA binding sites (K_d 11 nm and 500 μm, respectively) on human granulocytes (16). Most recently, nanomolar ABA has been shown to stimulate insulin secretion by human and murine pancreatic β cells and by rat insulinoma cell lines through a signaling pathway similar to the one described in human granulocytes (19). The autocrine release of ABA from glucose-stimulated human and rodent insulin-releasing cells, together with the fact that ABA is also produced by activated inflammatory cells, granulocytes (16), and monocytes (20), suggests that this hormone may contribute to the network of cytokine signals exchanged between inflammatory cells and pancreatic β cells, which is increasingly recognized as a fundamental mechanism in the development of the metabolic syndrome and type II diabetes (21–24).Based on (i) the sequence homology between the putative Arabidopsis ABA-receptor protein GCR2 and the human LANCL protein family, and (ii) the reported association of LANCL2 with the plasmamembrane, we investigated whether LANCL2 might be involved in ABA sensing in mammalian ABA-responsive cells. The results obtained indicate that LANCL2 is indeed, (i) required for ABA binding to the plasmamembrane of human granulocytes and (ii) necessary for the activation of the ABA signaling pathway, leading to the stimulation of the functional responses induced by ABA in human granulocytes and in rat insulinoma cells.     

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).     

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.The ultrastructure and physical properties of plant cell walls are known to be affected by boron deficiency (Kouchi and Kumazawa, 1976; Hirsch and Torrey, 1980; Fischer and Hecht-Buchholz, 1985; Matoh et al., 1992; Hu and Brown, 1994; Findeklee and Goldbach, 1996). Moreover, boron is predominantly localized in the cell wall when plants are grown with suboptimal boron (Loomis and Durst, 1991; Matoh et al., 1992; Hu and Brown, 1994; Hu et al., 1996). In radish, >80% of the cell wall boron is present in the pectic polysaccharide RG-II (Matoh et al., 1993; Kobayashi et al., 1996), which is now known to exist as a dimer that is cross-linked by a borate ester between two apiosyl residues (Kobayashi et al., 1996; O''Neill et al., 1996). Dimeric RG-II is unusually stable at low pH and is present in a large number of plant species (Ishii and Matsunaga, 1996; Kobayashi et al., 1996, 1997; Matoh et al., 1996; O''Neill et al., 1996; Pellerin et al., 1996; Kaneko et al., 1997). The widespread occurrence and conserved structure of RG-II (Darvill et al., 1978; O''Neill et al., 1990) have led to the suggestion that borate ester cross-linked RG-II is required for the development of a normal cell wall (O''Neill et al., 1996; Matoh, 1997).One approach for determining the function of boron in plant cell walls is to compare the responses to boron deficiency of growing plant cells that are dividing and synthesizing primary cell walls with those of growth-limited plant cells in which the synthesis of primary cell walls is negligible. Suspension-cultured cells are well suited for this purpose because they may be reversibly transferred from a growth phase to a stationary phase. Continuous cell growth phase is maintained by frequent transfer of the cells into new growth medium (King, 1981; Kandarakov et al., 1994), whereas a stationary cell population is obtained by feeding the cells with Suc and by not subculturing them. Cells in the stationary phase are characterized by mechanically stabilized primary walls and reduced biosynthetic activity. Here we describe the responses of suspension-cultured Chenopodium album L. cells in the growth and stationary phases to boron deficiency. These cells have a high specific-growth rate, no significant lag phase, and reproducible changes in their wall pore size during the transition from the growth phase to the stationary phase (Titel et al., 1997).     

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.Acinetobacter baumannii is an emerging opportunistic pathogen of increasing significance to health care institutions worldwide (1–3). The growing number of identified multiple drug resistant (MDR)¹ strains (2–4), the ability of isolates to rapidly acquire resistance (3, 4), and the propensity of this agent to survive harsh environmental conditions (5) account for the increasing number of outbreaks in intensive care, burn, or high dependence health care units since the 1970s (2–5). The burden on the global health care system of MDR A. baumannii is further exacerbated by standard infection control measures often being insufficient to quell the spread of A. baumannii to high risk individuals and generally failing to remove A. baumannii from health care institutions (5). Because of these concerns, there is an urgent need to identify strategies to control A. baumannii as well as understand the mechanisms that enable its persistence in health care environments.Surface glycans have been identified as key virulence factors related to persistence and virulence within the clinical setting (6–8). Acinetobacter surface carbohydrates were first identified and studied in A. venetianus strain RAG-1, leading to the identification of a gene locus required for synthesis and export of the surface carbohydrates (9, 10). These carbohydrate synthesis loci are variable yet ubiquitous in A. baumannii (11, 12). Comparison of 12 known capsule structures from A. baumannii with the sequences of their carbohydrate synthesis loci has provided strong evidence that these loci are responsible for capsule synthesis with as many as 77 distinct serotypes identified by molecular serotyping (11). Because of the non-template driven nature of glycan synthesis, the identification and characterization of the glycans themselves are required to confirm the true diversity. This diversity has widespread implications for Acinetobacter biology as the resulting carbohydrate structures are not solely used for capsule biosynthesis but can be incorporated and utilized by other ubiquitous systems, such as O-linked protein glycosylation (13, 14).Although originally thought to be restricted to species such as Campylobacter jejuni (15, 16) and Neisseria meningitidis (17), bacterial protein glycosylation is now recognized as a common phenomenon within numerous pathogens and commensal bacteria (18, 19). Unlike eukaryotic glycosylation where robust and high-throughput technologies now exist to enrich (20–22) and characterize both the glycan and peptide component of glycopeptides (23–25), the diversity (glycan composition and linkage) within bacterial glycosylation systems makes few technologies broadly applicable to all bacterial glycoproteins. Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26, 27) and the use of multiple fragmentation approaches (28, 29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30–33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought. For example, Nothaft et al. were able to show N-linked glycosylation was widespread in the Campylobacter genus and that two broad groupings of the N-glycans existed (34).During the initial characterization of A. baumannii O-linked glycosylation the use of selective enrichment of glycopeptides followed by mass spectrometry analysis with multiple fragmentation technologies was found to be an effective means to identify multiple glycosylated substrates in the strain ATCC 17978 (14). Interestingly in this strain, the glycan utilized for protein modification was identical to a single subunit of the capsule (13) and the loss of either protein glycosylation or glycan synthesis lead to decreases in biofilm formation and virulence (13, 14). Because of the diversity in the capsule carbohydrate synthesis loci and the ubiquitous distribution of the PglL O-oligosaccharyltransferase required for protein glycosylation, we hypothesized that the glycan variability might be also extended to O-linked glycosylation. This diversity, although common in surface carbohydrates such as the lipopolysaccharide of numerous Gram-negative pathogens (35), has only recently been observed within bacterial proteins glycosylation system that are typically conserved within species (36) and loosely across genus (34, 37).In this study, we explored the diversity within the O-linked protein glycosylation systems of Acinetobacter species. Our analysis complements the recent in silico studies of A. baumannii showing extensive glycan diversity exists in the carbohydrate synthesis loci (11, 12). Employing global strategies for the analysis of glycosylation, we experimentally demonstrate that the variation in O-glycan structure extends beyond the genetic diversity predicted by the carbohydrate loci alone and targets proteins of similar properties and identity. Using this knowledge, we developed a targeted approach for the detection of protein glycosylation, enabling streamlined analysis of glycosylation within a range of genetic backgrounds. We determined that; O-linked glycosylation is widespread in clinically relevant Acinetobacter species; inter- and intra-strain heterogeneity exist within glycan structures; glycan diversity, although extensive results in the generation of glycans with similar properties and that the utilization of a single glycan for capsule and O-linked glycosylation is a general feature of A. baumannii but may not be a general characteristic of all Acinetobacter species such as A. baylyi.     

Electric Signaling and Pin2 Gene Expression on Different Abiotic Stimuli Depend on a Distinct Threshold Level of Endogenous Abscisic Acid in Several Abscisic Acid-Deficient Tomato Mutants

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.The plant hormones ABA and JA play a predominant role in the conversion of environmental signals into changes in plant gene expression. An increase in endogenous ABA and JA levels precedes and is involved in Pin2 (proteinase inhibitor II) gene expression upon wounding (Peña-Cortés et al., 1989, 1991, 1995, 1996; Farmer and Ryan, 1992; Farmer et al., 1992). This increase in ABA and JA is not restricted to the tissue damaged directly but can also be detected in the nonwounded, systemically induced tissue (Peña-Cortés et al., 1989; Peña-Cortés and Willmitzer, 1995). The accumulation of ABA and JA have been described for several plant species, including potato, tomato, and tobacco (Sanchez-Serrano et al., 1991; Peña-Cortés and Willmitzer, 1995).Further evidence for the involvement of ABA and JA in wound-induced Pin2 gene expression was provided by a series of experiments in which potato plants were sprayed with ABA or JA and Pin2 mRNA accumulated in the absence of any wounding (Peña-Cortés et al., 1989; Hildmann et al., 1992). Both nonsprayed leaves and leaves that were sprayed directly showed increased Pin2 mRNA levels with a pattern identical to the one described for wounded plants (Peña-Cortés et al., 1988; Peña-Cortés and Willmitzer, 1995). Conclusive evidence for the involvement of ABA in wound-induced Pin2 activation was obtained from mutants impaired in ABA biosynthesis. Consequently, wound induction of Pin2 was not observed in the droopy mutant of potato or the sitiens mutant of tomato (Peña-Cortés et al., 1989). However, in these mutants treatment with ABA caused a return of the accumulation of Pin2 mRNA to levels normally found in wild-type plants upon wounding (Peña-Cortés et al., 1991).Like wounding, the application of electrical current was able to initiate ABA and JA accumulation in wild-type plants but not in ABA-deficient plants (Herde et al., 1996). These results suggested that, like wounding, electrical current requires the presence of ABA for the induction of Pin2 gene expression (Herde et al., 1996). In contrast to wounding and electrical current, burning of leaves activated Pin2 gene expression in sitiens mutants by directly triggering the biosynthesis of JA via an alternative pathway that is independent of endogenous ABA levels (Herde et al., 1996).To determine the endogenous levels of ABA that are sufficient to mediate electrical current-, heat-, and wound- induced Pin2 gene expression via electrical signals, we used several tomato mutants containing progressively reduced levels of ABA. The effects of these attenuated ABA levels on JA content and membrane potentials and the expression pattern of Pin2 genes were analyzed. Analysis of JA content was conducted to confirm the existence of an alternative pathway that is independent of endogenous ABA levels in the different ABA-deficient mutants.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.