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1.
Ilie Sachelaru Narcis Adrian Petriman Renuka Kudva Patrick Kuhn Thomas Welte Bettina Knapp Friedel Drepper Bettina Warscheid Hans-Georg Koch 《The Journal of biological chemistry》2013,288(23):16295-16307
Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements. 相似文献
2.
Hansj?rg G?tzke Isolde Palombo Claudio Muheim Elsa Perrody Pierre Genevaux Renuka Kudva Matthias Müller Daniel O. Daley 《The Journal of biological chemistry》2014,289(27):19089-19097
Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA. 相似文献
3.
Protein translocation across the endoplasmic reticulum membrane occurs via a "translocon" channel formed by the Sec61p complex. In yeast, two channels exist: the canonical Sec61p channel and a homolog called Ssh1p. Here, we used trapped translocation intermediates to demonstrate that a specific signal recognition particle-dependent substrate, Sec71p, is targeted exclusively to Ssh1p. Strikingly, we found that, in the absence of Ssh1p, precursor could be successfully redirected to canonical Sec61p, demonstrating that the normal targeting reaction must involve preferential sorting to Ssh1p. Our data therefore demonstrate that Ssh1p is the primary translocon for Sec71p and reveal a novel sorting mechanism at the level of the endoplasmic reticulum membrane enabling precursors to be directed to distinct translocons. Interestingly, the Ssh1p-dependent translocation of Sec71p was found to be dependent upon Sec63p, demonstrating a previously unappreciated functional interaction between Sec63p and the Ssh1p translocon. 相似文献
4.
The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Δ and yet3Δ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Δ or yet3Δ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins. 相似文献
5.
Thomas Becker Susanne E. Horvath Lena B?ttinger Natalia Gebert Günther Daum Nikolaus Pfanner 《The Journal of biological chemistry》2013,288(23):16451-16459
The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex. 相似文献
6.
Ke Wang Zhifen Yang Xu Liu Kai Mao Usha Nair Daniel J. Klionsky 《The Journal of biological chemistry》2012,287(45):37964-37972
Macroautophagy (hereafter autophagy) is a degradative cellular pathway that protects eukaryotic cells from stress, starvation, and microbial infection. This process must be tightly controlled because too little or too much autophagy can be deleterious to cellular physiology. The phosphatidylinositol (PtdIns) 3-kinase Vps34 is a lipid kinase that regulates autophagy, but the role of other PtdIns kinases has not been examined. Here we demonstrate a role for PtdIns 4-kinases and PtdIns4P 5-kinases in selective and nonselective types of autophagy in yeast. The PtdIns 4-kinase Pik1 is involved in Atg9 trafficking through the Golgi and is involved in both nonselective and selective types of autophagy, whereas the PtdIns4P 5-kinase Mss4 is specifically involved in mitophagy but not nonselective autophagy. Our data indicate that phosphoinositide kinases have multiple roles in the regulation of autophagic pathways. 相似文献
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8.
Denis G. Knyazev Alexander Lents Eberhard Krause Nicole Ollinger Christine Siligan Daniel Papinski Lukas Winter Andreas Horner Peter Pohl 《The Journal of biological chemistry》2013,288(25):17941-17946
In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. 相似文献
9.
Johannes H. Reithinger Chewon Yim Sungmin Kim Hunsang Lee Hyun Kim 《The Journal of biological chemistry》2014,289(22):15845-15855
The evolutionarily conserved Sec61 translocon mediates the translocation and membrane insertion of proteins. For the integration of proteins into the membrane, the Sec61 translocon opens laterally to the lipid bilayer. Previous studies suggest that the lateral opening of the channel is mediated by the helices TM2b and TM7 of a pore-forming subunit of the Sec61 translocon. To map key residues in TM2b and TM7 in yeast Sec61 that modulate lateral gating activity, we performed alanine scanning and in vivo site-directed photocross-linking experiments. Alanine scanning identified two groups of critical residues in the lateral gate, one group that leads to defects in the translocation and membrane insertion of proteins and the other group that causes faster translocation and facilitates membrane insertion. Photocross-linking data show that the former group of residues is located at the interface of the lateral gate. Furthermore, different degrees of defects for the membrane insertion of single- and double-spanning membrane proteins were observed depending on whether the mutations were located in TM2b or TM7. These results demonstrate subtle differences in the molecular mechanism of the signal sequence binding/opening of the lateral gate and membrane insertion of a succeeding transmembrane segment in a polytopic membrane protein. 相似文献
10.
Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β2-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments. 相似文献
11.
The two major components of the Eubacteria Sec-dependent protein translocation system are the heterotrimeric channel-forming component SecYEG and its binding partner, the SecA ATPase nanomotor. Once bound to SecYEG, the preprotein substrate, and ATP, SecA undergoes ATP-hydrolytic cycles that drive the stepwise translocation of proteins. Although a previous site-directed in vivo photocross-linking study (Mori, H., and Ito, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16159-16164) elucidated residues of SecY needed for interaction with SecA, no reciprocal study for SecA protein has been reported to date. In the present study we mapped residues of SecA that interact with SecY or SecG utilizing this approach. Our results show that distinct domains of SecA on two halves of the molecule interact with two corresponding SecY partners as well as with the central cytoplasmic domain of SecG. Our data support the in vivo relevance of the Thermotoga maritima SecA·SecYEG crystal structure that visualized SecYEG interaction for only one-half of SecA as well as previous studies indicating that SecA normally binds two molecules of SecYEG. 相似文献
12.
Jimena Castillo Bennett Carlos M. Roggero Franco E. Mancifesta Luis S. Mayorga 《The Journal of biological chemistry》2010,285(34):26269-26278
Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis. 相似文献
13.
The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgi-protein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and γ-tubulin and functioned in its centrosomal targeting. Both the N- and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis. 相似文献
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15.
Raunak Soman Jijun Yuan Andreas Kuhn Ross E. Dalbey 《The Journal of biological chemistry》2014,289(2):1023-1032
During membrane biogenesis, the M13 procoat protein is inserted into the lipid bilayer in a strictly YidC-dependent manner with both the hydrophobic signal sequence and the membrane anchor sequence promoting translocation of the periplasmic loop via a hairpin mechanism. Here, we find that the translocase requirements can be altered for PClep in a predictable manner by changing the polarity and charge of the peptide region that is translocated across the membrane. When the polarity of the translocated peptide region is lowered and the charged residues in this region are removed, translocation of this loop region occurs largely by a YidC- and Sec-independent mechanism. When the polarity is increased to that of the wild-type procoat protein, the YidC insertase is essential for translocation. Further increasing the polarity, by adding charged residues, switches the insertion pathway to a YidC/Sec mechanism. Conversely, we find that increasing the hydrophobicity of the transmembrane segments of PClep can decrease the translocase requirement for translocation of the peptide chain. This study provides a framework to understand why the YidC and Sec machineries exist in parallel and demonstrates that the YidC insertase has a limited capacity to translocate a peptide chain on its own. 相似文献
16.
Katsuhiko Kojima Yuji Amano Kazuhisa Yoshino Nobuyuki Tanaka Kazuo Sugamura Toshikazu Takeshita 《The Journal of biological chemistry》2014,289(48):33296-33310
The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process. 相似文献
17.
The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili. 相似文献
18.
Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation. 相似文献
19.
Annya M. Smyth Colin Rickman Rory R. Duncan 《The Journal of biological chemistry》2010,285(49):38141-38148
Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools. 相似文献
20.
Tomokazu Yamazaki Naoki Takata Matsuo Uemura Yukio Kawamura 《The Journal of biological chemistry》2010,285(30):23165-23176
The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C2A and C2B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C2A-C2B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C2B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins. 相似文献