A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

YidC mediates membrane protein insertion in bacteria

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.     

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61beta. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.     

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.     

Cotranslocational insertion of apolipoprotein B into the inner leaflet of the endoplasmic reticulum

Apolipoprotein (apo) B100 is required for the distribution of hepatic triglyceride to peripheral tissues as very-low-density lipoproteins. The translocation of apo B100 into the endoplasmic reticulum (ER) and its subsequent assembly into lipoprotein particles is of particular interest as the protein is both very large (relative molecular mass 512,000) and insoluble in water. It has been proposed that apo B translocation occurs in discrete stages and is completed post-translationally. Several sites of arrest of translocation were reported to be present in apo B15 (the N-terminal 15% of the protein). We have re-examined this question by in vitro translation coupled with translocation into microsomes, and find no evidence for transmembrane segments in truncated apo B proteins. Translocated apo B17 is strongly associated with the membrane of the ER, being only partially releasable with alkaline carbonate, and remaining bound to the microsomes following disruption with saponin. The efficient binding of short segments of apo B, despite the absence of transmembrane domains, suggests that apo B is cotranslationally inserted into the inner leaflet of the ER. This will obviate problems caused by the size and insolubility of apo B100, because the growing hydrophobic protein chains will never exist in a lipid-free form during translocation. From the inner leaflet, apo B in association with membrane-derived lipid can bud into the lumen of the ER to form nascent lipoprotein particles.     

Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway.

The transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems. Yeast genes encoding cytosolic components that participate in vesicular traffic were first identified from the collection of conditional-lethal sec-(secretory) mutants. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular membranes. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec7 protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with Sec7p-specific antibodies by immuno-isolation for biochemical and electron microscopic characterization. Sec7p on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies, implicate Sec7p as part of a (non-clathrin) vesicle coat. This Sec7p-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway.     

X-ray structure of a protein-conducting channel

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.     

Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.     

The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol.

In eukaryotic cells, incorrectly folded proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded by the proteasome. This pathway is co-opted by some viruses. For example, the US11 protein of the human cytomegalovirus targets the major histocompatibility complex class I heavy chain for cytosolic degradation. How proteins are extracted from the ER membrane is unknown. In bacteria and mitochondria, members of the AAA ATPase family are involved in extracting and degrading membrane proteins. Here we demonstrate that another member of this family, Cdc48 in yeast and p97 in mammals, is required for the export of ER proteins into the cytosol. Whereas Cdc48/p97 was previously known to function in a complex with the cofactor p47 (ref. 5) in membrane fusion, we demonstrate that its role in ER protein export requires the interacting partners Ufd1 and Npl4. The AAA ATPase interacts with substrates at the ER membrane and is needed to release them as polyubiquitinated species into the cytosol. We propose that the Cdc48/p97-Ufd1-Npl4 complex extracts proteins from the ER membrane for cytosolic degradation.     

Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage

Most eukaryotic secretory and membrane proteins insert co-translationally into the membrane of the rough endoplasmic reticulum (RER), and are targeted there by one or more NH2-terminal or internal signal sequences. However, little is known about the actual translocation and membrane integration processes. In particular, any energy requirements for targeting and integration have remained obscure because of the inability to uncouple the processes from concomitant protein synthesis. We recently showed that the human glucose transporter (GT), an integral membrane glycoprotein, can insert post-translationally into dog pancreatic microsomes with low but demonstrable efficiency in vitro, and that a fragment corresponding to the NH2-terminal 340 amino acids and 8 of the 12 membrane-spanning alpha-helixes of GT (GT-N) can insert with significantly greater efficiency. We report here that post-translational insertion of GT-N into pancreatic microsomes requires energy in the form of a phosphodiester bond, and suggest that co-translational insertion of proteins into the RER may also require energy independent of that used for polypeptide synthesis.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.     

Conformational transition of Sec machinery inferred from bacterial SecYE structures

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEbeta from the archaeon Methanococcus jannaschii, which lacks SecA. Here we present the 3.2-A-resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a 'pre-open' state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEbeta structure, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY. Molecular dynamics and disulphide mapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.     

In vitro synthesized bacterial outer membrane protein is integrated into bacterial inner membranes but translocated across microsomal membranes

The LamB protein is an integral membrane protein of the outer membrane of Escherichia coli. We have now found that, when synthesized in an E. coli cell-free translation system supplemented with inverted vesicles derived from the E. coli inner membrane, LamB protein is integrated into the vesicle membrane as assayed by its resistance to extraction at alkaline pH. These data suggest that the inner membrane is the primary site for integration of LamB protein prior to subsequent sorting to the outer membrane. When synthesized in a wheat germ cell-free translation system supplemented with canine microsomal membranes, LamB protein is glycosylated at one or two cryptic sites, and surprisingly, it is translocated across instead of being integrated into the vesicle membrane. We suggest that the translocation machinery of the microsomal membrane, although able to recognize the signal sequence(s) of LamB, is unable to recognize its stop-transfer sequence(s), thereby yielding translocation instead of integration.     

A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor.     

Three-dimensional structure of the bacterial protein-translocation complex SecYEG

Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.     

Preserving the membrane barrier for small molecules during bacterial protein translocation

Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids. While the cytoplasmic cavity of the channel is empty, the extracellular cavity is filled with a short helix called the plug, which moves out of the way during protein translocation. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial. Here, we have addressed this issue in intact Escherichia coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. We show that in the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained during protein translocation.     

Topological restriction of SNARE-dependent membrane fusion

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.     

人类新基因SEC15L3 cDNA的鉴定

从人胎脑cDNA文库中克隆到1条新的Sec15基因SEC15L3 cDNA,比已报道的SEC15L1(NM_019053)少4个外显子,并已提交GenBank,登录号为EF571007.RT-PCR和荧光定量PCR实验显示该基因在胰腺、脾脏、胸腺、前列腺和睾丸几个组织中表达量较高,而且与SEC15L1的组织表达谱有明显差异.生物信息学方法模拟SEC15L3蛋白,并与SEC15L1比较,结果显示两者有不同的高级结构,已推测出两者可能是不同exocyst的成分,并且在不同组织中行使不同功能.