Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition

Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.     

A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition

To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE). PE is required for the proper conformation and active transport function of LacY. The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa. Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport. These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures.     

Altering erythrocyte membrane composition with phospholipid exchange protein

• 1.1. In establishing a protocol for the use of a phospholipid (PL) 2 exchange protein (PLEP) preparation to incorporate fluorescent lipids into membrane fragments, the potential for the PLEP preparation to alter membrane composition was investigated.
• 2.2. Cholesterol was removed from erythrocyte ghosts and PL content was increased after incubation of the membranes with phosphatidylcholine (PC) vesicles and a nonspecific bovine liver PLEP preparation.
• 3.3. The elevated PL/cholesterol ratio of the ghosts decreased fluorescence polarization of diphenylhexatriene (DPH), indicating an increased average membrane fluidity.
• 4.4. The increased PL/cholesterol ratio and reduced DPH fluorescence polarization were prevented by including cholesterol in the PC vesicles during the initial incubation of ghosts with PLEP preparation and lipid.
• 5.5. In addition, the PC content of ghost membranes was elevated after incubation with PLEP preparation and either PC or mixed PC-cholesterol vesicles.

     

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)‐domains associated with diverse ER‐organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1‐enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1‐deficient mammalian cells and other model systems.      

Non-alpha-helical elements modulate polytopic membrane protein architecture

In "all alpha-fold" transmembrane proteins, including ion channels, G-protein-coupled receptors (GPCRs), bacterial rhodopsins and photosynthetic reaction centers, relatively long alpha-helices, straight, curved or kinked, pack into compact elliptical or circular domains. Using both existing and newly developed tools to analyze transmembrane segments of all available membrane protein three-dimensional structures, including that very recently elucidated for the GPCR, rhodopsin, we report here the finding of frequent non-alpha-helical components, i.e. 3(10)-helices ("tight turns"), pi-helices ("wide turns") and intrahelical kinks (often due to residues other than proline). Often, diverse helical types and kinks concatenate over long segments and produce complex inclinations of helical axis, and/or diverse frame shifts in the "canonical", alpha-helical side-chain pattern. Marked differences in transmembrane architecture exist even between seemingly structurally related proteins, such as bacteriorhodopsin and rhodopsin. Deconvolution of these non-canonical features into their composite elements is essential for understanding the pleiotropy of polytopic protein structure and function, and must be considered in developing valid macromolecular models.     

Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis.

Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity [Braun, P., Persson, B., Kaback, H. R., and von Heijne, G. (1997) J. Biol. Chem. 272, 29566-29571]. Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli. Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation. Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices. In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions. More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively.     

Endoplasmic reticulum-associated degradation of a degron-containing polytopic membrane protein

Abstract

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.     

Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.     

Signal peptidase can cleave inside a polytopic membrane protein

The signal peptides of most proteins targeted to the endoplasmic reticulum are specifically cleaved by signal peptidase. Although potential cleavage sites occur frequently in polytopic proteins after membrane-spanning segments, processing is restricted to the first hydrophobic domain, suggesting that signal peptidase might not have access to subsequently translocated, internal domains. To test this hypothesis, we replaced the third transmembrane segment of an artificial threefold membrane-spanning protein by a sequence which is normally an amino-terminal signal. Upon in vitro translation and insertion into microsomes, efficient cleavage at this sequence was observed, thus demonstrating the ability of signal peptidase to cleave within polytopic membrane proteins.     

Dissecting the ER-associated degradation of a misfolded polytopic membrane protein

It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p(*), a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p(*) association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.     

Conserved ERAD-like quality control of a plant polytopic membrane protein

The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism.     

The ammonia channel protein AmtB from Escherichia coli is a polytopic membrane protein with a cleavable signal peptide

The Escherichia coli ammonia channel protein, AmtB, is a homotrimeric polytopic inner membrane protein in which each subunit has 11 transmembrane helices. We have shown that the structural gene amtB encodes a preprotein with a signal peptide that is cleaved off to produce a topology with the N-terminus in the periplasm and the C-terminus in the cytoplasm. Deletion of the signal peptide coding region results in significantly lower levels of AmtB accumulation in the membrane but modification of the signal peptidase cleavage site, leading to aberrant cleavage, does not prevent trimer formation and does not inactivate the protein. The presence of a signal peptide is apparently not a conserved feature of all prokaryotic Amt proteins. Comparison of predicted AmtB sequences suggests that while Amt proteins in Gram-negative organisms utilize a signal peptide, the homologous proteins in Gram-positive organisms do not.     

F-box protein specificity for g1 cyclins is dictated by subcellular localization

Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4) and SCF(Grr1), redundantly target Cln3 for degradation. While the F-box proteins (FBPs) Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.     

Inhibition of Friend cell erythrodifferentiation by modification of membrane phospholipid composition by choline analogues

Dimethylsulfoxide-stimulated Friend leukemia cell erythrodifferentiation was inhibited by choline analogues such as N-monomethylethanolamine and N,N-dimethylethanolamine. Phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine were then accumulated in the cell membranes. N-Monomethylethanolamine also inhibited Friend leukemia cell erythrodifferentiation stimulated by hexamethylene bisacetamide and N-methylacetamide, but did not inhibit differentiation induced by sodium butyrate. This inhibitory effect of N-monomethylethanolamine was partially abrogated by spermine.     

Time scale of protein aggregation dictated by liquid-liquid demixing

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point-mutant form of normal human Hemoglobin (HbA), is the first recognized and best-studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy-HbS aggregates by referring them to the (concentration-dependent) temperature T(s) characterizing the occurrence of the phase transition of liquid-liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter epsilon = (T - T(s)) / T(s), familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter-protein interactions leading to nucleation. Referring to epsilon shows how in a narrow temperature span, that is at T - T(s), nucleation kinetics can undergo orders-of-magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering T(s) and consequently epsilon. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases.     

Prostate epithelial differentiation is dictated by its surrounding stroma

Prostate epithelial differentiation is dictated by its surrounding stroma which determines androgen induced growth responsiveness and expression of specific secretory proteins in normal prostate gland. During neoplastic progression, organ specific stroma has been shown to determine the rate of neoplastic progression from androgen-dependent to androgen-independent and metastatic states. Although growth factors and extracellular matrix are recognized as important contributors to prostate epithelial growth, hormonal responsiveness, and neoplastic progression, the exact mechanism of intercellular communication between stromal and epithelial cells remains undefined. In addition to the importance of defining the reciprocal interaction between stromal and epithelial interaction in the prostate, clonal interaction between two dissimilar prostate epithelial cells is also recognized to contribute to disease progression. In this review, we summarized recent advances made in delineating molecular mechanisms underlying stromal epithelial interaction and clonal interaction between androgen-dependent and androgen-independent prostate cancer cells in vivo and in culture. Understanding cellular interaction between prostate epithelium and its surrounding stroma could help us in developing metastatic models of prostate carcinogenesis. This concept will allow us to define epithelial-specific markers, markers induced as the result of stromal-epithelial interaction, and stroma-associated markers. These markers together will assist us in diagnosing, preventing, prognosing and treating prostate cancer more efficaciously in the future.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

Bacterial senescence: protein oxidation in non-proliferating cells is dictated by the accuracy of the ribosomes

We have investigated the causal factors behind the age-related oxidation of proteins during arrest of cell proliferation. A proteomic approach demonstrated that protein oxidation in non-proliferating cells is observed primarily for proteins being produced in a number of aberrant isoforms. Also, these cells exhibited a reduced translational fidelity as demonstrated by both proteomic analysis and genetic measurements of nonsense suppression. Mutants harboring hyperaccurate ribosomes exhibited a drastically attenuated protein oxidation during growth arrest. In contrast, oxidation was augmented in mutants with error-prone ribosomes. Oxidation increased concomitantly with a reduced rate of translation, indicating that the production of aberrant, and oxidized proteins, is not the result of titration of the co-translational folding machinery. The age-related accumulation of the chaperones, DnaK and GroEL, was drastically attenuated in the hyperaccurate rpsL mutant, demonstrating that the reduced translational fidelity in growth-arrested cells may also be a primary cause for the induction of the heat shock regulon. The data point to an alternative way of approaching the causal factors involved in protein oxidation in eukaryotic G(0) cells.