Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus

Using a cytosol and nucleotide dependent assay that we previously developed, we have investigated the requirement for coat proteins in the in vitro production of trans-Golgi network (TGN)-derived vesicles from a Madin-Darby canine kidney (MDCK) cell Golgi fraction that contains the ³⁵S-labeled, terminally glycosylated, envelope glycoprotein of vesicular stomatitis virus (VSV-G) accumulated in the TGN. We found that the TGN-derived vesicles, like those involved in intra-Golgi transport and in retrograde transport to the endoplasmic reticulum, contain a coatomer coat and that coatomer is required for their formation. Thus, after they are produced with GTPγS, the coated vesicles could be captured on beads containing anticoatomer antibody. Moreover, a cytosolic protein fraction depleted of coatomer could not support vesicle formation but it did so after purified coatomer was added. We also determined that P200/myosin II does not play an essential role in the in vitro generation of TGN-derived vesicles. Thus, removal of this protein from the cytosol, by differential salt precipitation or binding to phalloidin-induced actin filaments, had no effect on vesicle generation. Nevertheless, immunodepletion of cytosol using the anti-P200/myosin II AD7 antibody abolished vesicle generation and that antibody was capable of effectively immunocapturing coated vesicles, even when these were generated in the absence of P200/myosin II. These effects, however, are explained by the unexpected finding that the AD7 antibody interacts with undenatured coatomer.     

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

The current diffusion-retention model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding to nuclear proteins or DNA. Because some data indicate that the sorting of baculovirus envelope proteins to the INM is protein-mediated, we have examined the early stages of INM protein integration and sorting by using photocrosslinking. Both viral and host INM-directed proteins were integrated cotranslationally through the endoplasmic reticulum translocon, and their nonrandom photocrosslinking to two translocon proteins, Sec61alpha and translocating chain-associated membrane protein (TRAM), revealed that the first transmembrane sequence (TMS) of each viral and host INM-directed protein occupied a very similar location within the translocon. Because few TMSs of non-INM-directed membrane proteins photocrosslink to TRAM, it seems that the INM-directed TMSs occupy different sites within the translocon than do non-INM-directed TMSs. The distinct proximities of translocon components to INM-directed TMSs strongly suggest that such TMSs are recognized and initially sorted within the translocon. Taken together, these data indicate that membrane protein sorting to the INM is an active process involving specific nonnuclear proteins.     

Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae.

Protein and peptide export from the Saccharomyces cerevisiae endoplasmic reticulum was examined in vitro using the secretory protein pro-alpha-factor and a synthetic tripeptide containing the acceptor site for N-linked glycosylation as substrates. The release of both glycosylated pro-alpha-factor and glycotripeptide from the endoplasmic reticulum was dependent on cytosol, temperature, and ATP. Antibodies against two proteins essential for the formation of transport vesicles, Sec23p and p105, inhibited glyco-pro-alpha-factor exit from the endoplasmic reticulum but did not affect the release of the glycosylated tripeptide. Furthermore, in contrast to pro-alpha-factor, the exported glycopeptide was not associated with a membrane fraction and did not acquire Golgi-specific alpha(1-6)-linked mannose residues. We conclude that the glycosylated tripeptide leaves the yeast endoplasmic reticulum by a route different from the secretory pathway, possibly through an ATP-driven pump.     

Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex A alpha A beta dimers from the endoplasmic reticulum/cis-Golgi compartment.

Invariant chain (Ii) is a nonpolymorphic, non-major histocompatibility complex (MHC)-encoded glycoprotein that rapidly associates with newly synthesized class II MHC alpha and beta chains in the rough endoplasmic reticulum. This oligomerization of Ii, alpha, and beta and their cotransport within the cell led to speculation that Ii was an essential alpha beta transport protein. However, direct tests failed to show an absolute requirement for Ii in class II MHC molecule transport. More recently, it has become clear that different class II alpha beta chain combinations vary greatly in their efficiency of cell-surface expression, based largely on the allelic origin of the alpha and beta amino-terminal regions. Because the previous tests of Ii for a role in class II molecule expression utilized efficiently expressed alpha beta combinations, we have reexamined this question with several haplotype-mismatched murine A alpha and A beta chain combinations of various potentials for cell-surface expression. Using a transient expression assay in Ii-negative COS cells, we find that many inefficiently expressed alpha beta combinations show marked augmentation of surface expression upon cosynthesis of Ii. This effect is absent or minimal with evolutionarily coselected, haplotype-matched chains that give efficient expression alone. Biochemical studies show that at least one component of the Ii effect is an increased egress of already formed alpha beta dimers from the rough endoplasmic reticulum/cis-Golgi. We suggest that these results reflect the interaction of Ii with the peptide-binding domain of the poorly expressed class II molecules, either aiding in maintenance of a transportable conformation or competing with endoplasmic reticulum retention proteins, and thus enhancing movement to the cell surface. These results suggest a complex and variable role for trans-associated alpha and beta chains in the immune responses of MHC heterozygotes and provide a method for examining Ii interaction with class II MHC molecules independent of measurement of peptide presentation to T cells.     

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.     

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.     

Identification and characterization of COPIa- and COPIb-type vesicle classes associated with plant and algal Golgi

Coat protein I (COPI) vesicles arise from Golgi cisternae and mediate the recycling of proteins from the Golgi back to the endoplasmic reticulum (ER) and the transport of Golgi resident proteins between cisternae. In vitro studies have produced evidence for two distinct types of COPI vesicles, but the in vivo sites of operation of these vesicles remain to be established. We have used a combination of electron tomography and immunolabeling techniques to examine Golgi stacks and associated vesicles in the cells of the scale-producing alga Scherffelia dubia and Arabidopsis preserved by high-pressure freezing/freeze-substitution methods. Five structurally distinct types of vesicles were distinguished. In Arabidopsis, COPI and COPII vesicle coat proteins as well as vesicle cargo molecules (mannosidase I and sialyltransferase-yellow fluorescent protein) were identified by immunogold labeling. In both organisms, the COPI-type vesicles were further characterized by a combination of six structural criteria: coat architecture, coat thickness, membrane structure, cargo staining, cisternal origin, and spatial distribution. Using this multiparameter structural approach, we can distinguish two types of COPI vesicles, COPIa and COPIb. COPIa vesicles bud exclusively from cis cisternae and occupy the space between cis cisternae and ER export sites, whereas the COPIb vesicles bud exclusively from medial- and trans-Golgi cisternae and are confined to the space around these latter cisternae. We conclude that COPIa vesicle-mediated recycling to the ER occurs only from cis cisternae, that retrograde transport of Golgi resident proteins by COPIb vesicles is limited to medial and trans cisternae, and that diffusion of periGolgi vesicles is restricted.     

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

Peptides and misfolded secretory proteins are transported efficiently from the endoplasmic reticulum (ER) lumen to the cytosol, where the proteins are degraded by proteasomes. Protein export depends on Sec61p, the ribosome-binding core component of the protein translocation channel in the ER membrane. We found that prebinding of ribosomes abolished export of a glycopeptide from yeast microsomes. Deletion of SSH1, which encodes a ribosome-binding Sec61p homologue in the ER, had no effect on glycopeptide export. A collection of cold-sensitive sec61 mutants displayed a variety of phenotypes: two mutants strongly defective in misfolded protein export from the ER, sec61-32 and sec61-41, displayed only minor peptide export defects. Glycopeptide export was severely impaired, however, in several sec61 mutants that were only marginally defective in misfolded protein export. In addition, a mutation in SEC63 strongly reduced peptide export from the ER. ER-luminal ATP was required for both misfolded protein and glycopeptide export. We conclude that the protein translocation channel in the ER membrane mediates glycopeptide transport across the ER membrane.     

Exiting the endoplasmic reticulum

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.     

Sec53, a protein required for an early step in secretory protein processing and transport in yeast, interacts with the cytoplasmic surface of the endoplasmic reticulum.

The sec53 mutant is a conditional lethal yeast secretory mutant. At 37 degrees C, precursors to exported proteins become firmly attached to the endoplasmic reticulum membrane and are not released into the lumen in a soluble form. The accumulated precursors are insoluble in the detergent Triton X-100; however, urea, a known protein denaturant, solubilizes them. Using antibody directed against the Sec53 protein, we found that a substantial portion of the Sec53 protein is associated with the cytoplasmic surface of the endoplasmic reticulum membrane. Membrane-bound Sec53 protein is largely insoluble in Triton X-100, but the protein is effectively released from the membrane by urea. We propose that the Sec53 protein may be a member of a complex of proteins required for an early step in protein processing and transport.     

Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65.

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.     

The COPII pathway and hematologic disease

Multiple diseases, hematologic and nonhematologic, result from defects in the early secretory pathway. Congenital dyserythropoietic anemia type II (CDAII) and combined deficiency of coagulation factors V and VIII (F5F8D) are the 2 known hematologic diseases that result from defects in the endoplasmic reticulum (ER)-to-Golgi transport system. CDAII is caused by mutations in the SEC23B gene, which encodes a core component of the coat protein complex II (COPII). F5F8D results from mutations in either LMAN1 (lectin mannose-binding protein 1) or MCFD2 (multiple coagulation factor deficiency protein 2), which encode the ER cargo receptor complex LMAN1-MCFD2. These diseases and their molecular pathogenesis are the focus of this review.     

葡萄糖调节蛋白78在非酒精性脂肪性肝病发病中的作用*

葡萄糖调节蛋白78（GRP78）作为一个重要的内质网分子伴侣,在蛋白质的折叠、转运和内质网应激反应过程中发挥重要作用。GRP78还存在于肿瘤细胞、内皮细胞和单核细胞的表面,可作为病毒进入宿主细胞的受体。内质网应激是非酒精性脂肪性肝病(NAFLD)的一个重要的发病机制。对GRP78功能的深入挖掘,将为探索NAFLD的防御机制提供新思路。     

Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.Most of the transmembrane secretory cargo proteins from the endoplasmic reticulum (ER) are selectively exported in cytosolic coat protein complex II (COPII) vesicles via direct interaction of their export motif with the COPII coat. The COPII coat core machinery consists of five cytosolic proteins: Sar1, Sec23, Sec24, Sec13, and Sec31 (secretory pathway proteins) (1). Sec24 is considered to be the primary subunit responsible for binding to membrane cargo proteins at the ER and concentrating them into the forming vesicle (2). Some of these cargo proteins require the assistance of cytosolic or membrane-spanning accessory adaptor proteins for their incorporation into COPII vesicles. Several adaptor proteins have been identified to assist the COPII machinery in yeast (3–5); however, fewer have been characterized in higher eukaryotes. In metazoans, ERGIC-53 mediates the export of blood clotting factors, Cathepsin Z and C and α-1 antitrypsin (6), and SCAP [sterol-regulatory elementary binding protein (SREBP) cleavage activating protein] mediates the regulated transport of SREBP protein from the ER to the Golgi in cells that are sterol-deficient (7). Most COPII adaptor proteins are membrane-embedded, but at least one example of a cytosolic accessory protein, 14-3-3, has been proposed to control the anterograde trafficking of many of cell surface receptor proteins, possibly at the level of the ER (8). 14-3-3s are small (30 kDa), acidic, and ubiquitously expressed eukaryotic proteins that are conserved from yeast to mammals and modulate various cellular processes by interacting with a variety of target proteins (9, 10). These include cell cycle regulation, signaling by MAP kinases, apoptosis, and transfer of signaling molecules between the nucleus and cytosol (11–14). Yeast cell viability depends on the expression of at least one of the two 14-3-3 isoforms (Bmh1 and Bmh2) (15). There are seven different isoforms in mammals (β, γ, δ, ε, η, σ, θ), some of which show differential tissue localization (14). Because of their redundant roles in cellular processes, depleting cellular levels of 14-3-3 to study a particular process poses a challenge. It is thought that their role in trafficking is to interfere with the ER retention/retrieval motif of target membrane proteins, and thus promote the transport of these cargos to the cell surface (16). For some proteins (e.g., KCNK3 and MHC class II, GPR15) (17–19), recruitment of 14-3-3 requires phosphorylation of a residue involved in 14-3-3 binding, whereas in other proteins (e.g., Kir6.2) 14-3-3 recognizes the correct assembly of multimeric proteins (20, 21).In this paper we examine the role of 14-3-3 proteins as an adaptor for COPII vesicular transport of SAC1 (suppressor of actin mutations 1-like protein). SAC1 is a phosphatidyl inositol-4 (PI4) lipid phosphatase that belongs to a family of enzymes with a CX₅R(T/S) Sac catalytic domain, which is conserved from yeast to metazoans. Sac proteins control several cellular processes, including phosphoinositide homeostasis, membrane trafficking, and cytoskeleton organization. SAC1 is a 587-aa transmembrane protein with both N- and C-terminal domains exposed to the cytosol. Deletion of SAC1 in yeast and mammalian cells leads to changes in Golgi morphology and function and a SAC1 mouse knockout is embryonically lethal. Recently, SAC1 has been identified as Drosophila vesicle-associated protein binding partner and down-regulation of Drosophila vesicle-associated protein or SAC1 in Drosophila leads to the pathogenesis associated with amyotrophic lateral sclerosis (22).It has been reported previously that SAC1 is localized to the Golgi membranes only when cells are starved for nutrients or growth factors, but remains in the ER under normal growth conditions (23, 24). Given the role for PI(4)P in vesicle traffic from the trans Golgi network, starvation conditions that lodge SAC1 and thus deplete the local supply of PI(4)P in the Golgi may suppress anterograde traffic in cells that must cease net cell growth. The regulation of SAC1 traffic may be crucial to the control of cell growth and anterograde membrane traffic.The retrieval of mammalian SAC1 from the Golgi to the ER in the presence of growth factors or mitogens is controlled by COPI-mediated retrograde transport and requires the p38 MAPK pathway (23). Although the regulation of SAC1 retrieval from the Golgi has been reported, little is known about the control of SAC1 export from the ER under conditions of serum starvation. Recently, the N-terminal cytoplasmic domain of SAC1 was reported to contribute to Golgi localization in mammalian cells (25). We have established a cell-free reconstitution system that recapitulates the biogenesis and ER export of SAC1 and identified 14-3-3 proteins as an important factor in the packaging of SAC1 into COPII transport vesicles. Given the role of 14-3-3 proteins in various signaling pathways and the fact that SAC1 transport is affected by the p38 MAPK pathway, an understanding of the molecular role of 14-3-3 proteins in vesicular traffic could provide a mechanistic link between signaling and membrane assembly (23).     

Deletion of 3 residues from the C-terminus of MCFD2 affects binding to ERGIC-53 and causes combined factor V and factor VIII deficiency

Combined factor V and factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder. F5F8D is genetically linked to mutations in the transmembrane lectin ERGIC-53 and its soluble interaction partner MCFD2. The ERGIC-53/MCFD2 protein complex functions as transport receptor of coagulation factors V and VIII by mediating their export from the endoplasmic reticulum (ER). Here, we studied a F5F8D patient who was found to be a compound heterozygote for 2 novel mutations in MCFD2: a large deletion of 8.4 kb eliminating the 5'UTR of the gene and a nonsense mutation resulting in the deletion of only 3 amino acids (DeltaSLQ) from the C-terminus of MCFD2. Biochemical and structural analysis of the DeltaSLQ mutant demonstrated impaired binding to ERGIC-53 due to modification of the 3-dimensional structure of MCFD2. Our results highlight the importance of the ERGIC-53/MCFD2 protein interaction for the efficient secretion of coagulation factors V and VIII.     

Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins

Two classes of sterols, cholesterol and oxysterols, block export of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi by preventing the binding of COPII-coated proteins to a hexapeptide sorting signal (MELADL) in Scap, the SREBP-escort protein. Here, we show that anti-MELADL blocks COPII binding in vitro, and microinjection of Fab anti-MELADL blocks Scap.SREBP movement in cells. Cholesterol and oxysterols block COPII binding to MELADL by binding to different intracellular receptors, cholesterol to Scap and oxysterols to Insig. Cysteine labeling shows that both binding events produce a conformational change near the MELADL sequence, abrogating COPII binding but not anti-MELADL binding. Mutagenesis experiments raise the possibility that the distance of MELADL from the ER membrane is crucial for COPII binding, and we speculate that sterols and Insig block SREBP transport by altering the location of MELADL with respect to the membrane, rendering it inaccessible to COPII proteins.     

Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.     

"BFA bodies": a subcompartment of the endoplasmic reticulum.

A specialized region of the endoplasmic reticulum--the BFA body--is defined by the site of accumulation of coatomer when nonclathrin coat protein (COP)-coated vesicle assembly is prevented by the drug brefeldin A (BFA). BFA bodies are formed by part smooth, part rough domains of endoplasmic reticulum that are cis to the classical transitional endoplasmic reticulum and to BFA-induced Golgi remnants.