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1.
During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.  相似文献   

2.
In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.  相似文献   

3.
We have developed a non-steady-state mathematical model describing post-translational protein translocation across the endoplasmic reticulum membrane. Movement of the polypeptide chain through the channel in the endoplasmic reticulum membrane is considered to be a stochastic process which is biased at the lumenal side of the channel by the binding of BiP (Kar2p), a member of the Hsp70 family of ATPases (ratcheting model). Assuming that movement of the chain through the channel is caused by passive diffusion (Brownian ratchet), the model describes all available experimental data. The optimum set of model parameters indicates that the ratcheting mechanism functions at near-maximum rate, being relatively insensitive to variations of the association or dissociation rate constants of BiP or its concentration. The estimated rate constant for diffusion of a polypeptide inside the channel indicates that the chain makes contact with the walls of the channel. Since fitting of the model to the data required that the backward rate constant be larger than the forward constant during early diffusion steps, translocation must occur against a force. The latter may arise, for example, from the unfolding of the polypeptide chain in the cytosol. Our results indicate that the ratchet can transport polypeptides against a free energy of about 25 kJ/mol without significant retardation of translocation. The modeling also suggests that the BiP ratchet is optimized, allowing fast translocation to be coupled with minimum consumption of ATP and rapid dissociation of BiP in the lumen of the ER. Finally, we have estimated the maximum hydrophobicity of a polypeptide segment up to which lateral partitioning from the channel into the lipid phase does not result in significant retardation of translocation.  相似文献   

4.
Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.  相似文献   

5.
The mitochondrial protein import motor   总被引:2,自引:0,他引:2  
Strub A  Lim JH  Pfanner N  Voos W 《Biological chemistry》2000,381(9-10):943-949
Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.  相似文献   

6.
W Mothes  S Prehn    T A Rapoport 《The EMBO journal》1994,13(17):3973-3982
We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.  相似文献   

7.
Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.  相似文献   

8.
On the role of subunit III in proton translocation in cytochromec oxidase   总被引:7,自引:0,他引:7  
Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,M r 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.  相似文献   

9.
During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.  相似文献   

10.
We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.  相似文献   

11.
We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behaviour of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane.  相似文献   

12.
The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.  相似文献   

13.
《The Journal of cell biology》1986,103(6):2253-2261
The requirement for ribonucleotides and ribonucleotide hydrolysis was examined at several distinct points during translocation of a secretory protein across the endoplasmic reticulum. We monitored binding of in vitro-assembled polysomes to microsomal membranes after removal of ATP and GTP. Ribonucleotides were not required for the initial low salt- insensitive attachment of the ribosome to the membrane. However, without ribonucleotides the nascent secretory chains were sensitive to protease digestion and were readily extracted from the membrane with either EDTA or 0.5 M KOAc. In contrast, nascent chains resisted extraction with either EDTA or 0.5 M KOAc and were insensitive to protease digestion after addition of GTP or nonhydrolyzable GTP analogues. Translocation of the nascent secretory polypeptide was detected only when ribosome binding was conducted in the presence of GTP. Thus, translocation-competent binding of the ribosome to the membrane requires the participation of a novel GTP-binding protein in addition to the signal recognition particle and the signal recognition particle receptor. The second event we examined was translocation and processing of a truncated secretory polypeptide. Membrane-bound polysomes bearing an 86-residue nascent chain were generated by translation of a truncated preprolactin mRNA. Ribonucleotide- independent translocation of the polypeptide was detected by cleavage of the 30-residue signal sequence after puromycin termination. Nascent chain transport, per se, is apparently dependent upon neither ribonucleotide hydrolysis nor continued elongation of the polypeptide once a functional ribosome-membrane junction has been established.  相似文献   

14.
SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.  相似文献   

15.
A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time.  相似文献   

16.
Mitra K  Frank J 《FEBS letters》2006,580(14):3353-3360
The protein-conducting channel (PCC) must allow both the translocation of soluble polypeptide regions across, and the lateral partitioning of hydrophobic transmembrane helices (TMHs) into, the membrane. We have analyzed existing structures of ribosomes and ribosome-PCC complexes and observe conformational changes suggesting that the ribosome may sense and orient the nascent polypeptide and also facilitate conformational changes in the PCC, subsequently directing the nascent polypeptide into the appropriate PCC-mediated translocation mode. The PCC is predicted to be able to accommodate one central, consolidated channel or two segregated pores with different lipid accessibilities, which may enable the lipid-mediated partitioning of a TMH from one pore, while the other, aqueous, pore allows translocation of a hydrophilic polypeptide segment. Our hypothesis suggests a plausible mechanism for the transitioning of the PCC between different configurations.  相似文献   

17.
The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.  相似文献   

18.
In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in?vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the "posttranslational" translocation pathway.  相似文献   

19.
Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.  相似文献   

20.
Nascent polypeptide chains synthesized by membrane bound ribosomes are cotranslationally translocated through and integrated into the endoplasmic reticulum translocon. Hydrophobic segments and positive charges on the chain are critical to halt the ongoing translocation. A marginally hydrophobic segment, which cannot be inserted into the membrane by itself, can be a transmembrane segment depending on its downstream positive charges. In certain conditions, positive charges even 60 residues downstream cause the marginally hydrophobic segment to span the membrane by inducing the segment to slide back from the lumen. Here we systematically examined the effect of a core sugar chain on the fate of a marginally hydrophobic segment using a cell-free translation and translocation system. A sugar chain added within 12 residues upstream of the marginally hydrophobic segment prevents the sliding back and promotes forward movement of the polypeptide chain. The sugar chain apparently functions as a ratchet to keep the polypeptide chain in the lumen. We propose that the sugar chain is a third topology determinant of membrane proteins, in addition to a hydrophobic segment and positive charges of the nascent chain.  相似文献   

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