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.

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1) upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain kappa (NS1) which is unfolded in unassembled molecules. We investigated the fate of kappa (NS1) glycosylated at position 70 (kappa 70) and of a double mutant (kappa 18/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappa (NS1) chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.

We identified a human cDNA sequence encoding a polypeptide of 760 amino acids that shares 53% homology and 25.6% identity with the yeast DnaJ-like endoplasmic reticulum (ER) translocon component Sec63p. Three epitope-specific antisera revealed a protein of an apparent molecular mass of 83 kDa, both in human cell extracts and in dog pancreatic microsomes. Biochemical analyses show that it is an integral membrane protein of the rough ER, which has the DnaJ domain located in the ER lumen. The novel Sec63 protein could thus represent a key component of the mammalian ER protein translocation machinery.

To study the degradation requirements of unassembled immunoglobulin (Ig) chains, we heterologously expressed a cDNA encoding the secretory form of murine mu in the yeast S. cerevisiae, We found that mu chains were translocated into and retained in the endoplasmic reticulum (ER) as they were N-glycosylated and bound to the yeast homolog of BIP, Kar2p. Similar to mutant yeast carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu protein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6p and Ubc7p or in cells overexpressing mutant ubiquitin, Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the accumulation of polyubiquitinated mu chains that were still glycosylated. By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step. In contrast to the situation for CPY*, the ER-transmembrane protein Der1p is not essential far mu degradation. Strikingly, however, the CHX-induced accumulation of polyubiquitinated Ig mu, chains was stronger in Delta der1-mutants as compared to wild-type cells, indicating an additive effect of two inhibitory conditions. The results support a previously unknown activity of CHX, i.e. impairing the degradation of transport-incompetent secretory mu chains. Moreover, this activity wilt allow to dissect substrate-specific steps in ER associated protein degradation.

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER), Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, kappa(NS1) and lambda(FS62), are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells, Furthermore, the BiP/lambda(FS62) Ig L chain complex appears to be more stable than the BiP/kappa(NS1) complex, Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of kappa(NS1) and lambda(FS62) Ig L chains, Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains, Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.

A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the K-d heavy chain of mouse MHC class I, SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2K(d) molecules to the plasma membrane was only slightly affected, This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man(5)GlcNAc(2), which was monoglucosylated in the protein-bound form, Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2K(d) were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport, The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion, The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue, Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation.

Oligomeric proteins usually have to assemble into their final quartenary structure to be secreted. However, most immunoglobulin (Ig) light (L) chains can be exported as free chains, whereas only a few Ig L chains, here referred to as export-incompetent, have to assemble with Ig heavy (H) chains into antibody molecules to be secreted. In the absence of Ig H chain expression, these export-incompetent Ig L chains remain bound to BiP as partially folded monomers with only one of the two internal disulfide bonds being formed. To understand the apparent discrepancy in Ig L chain export, we performed assembly studies with chimeric Ig chains and found that the variable (V) domain of the export-incompetent NS1 kappa chain cannot mediate homodimer formation. Conversely, the V domain of the export-competent J558L lambda1 chain supports homodimer formation and, concordantly, these Ig L chains are secreted as noncovalently or covalently linked homodimers. We show that the export-incompetent mutant lambda1 FS62 chain forms disulfide bonds in both domains only upon pairing with Ig H chain and is secreted as part of an antibody. Therefore, Ig L chain assembly seems to be a prerequisite for complete folding, indicating that Ig L chain secretion generally depends on either homo- or heterodimer formation. We discuss a mechanism that controls oligomerization by monitoring the conformation of individual subunits that cannot proceed in folding prior to successful assembly.

The first constant domain (CH1) of immunoglobulin heavy (H) chains is essential for BiP-mediated retention of unassembled H chains in the endoplasmic reticulum (ER). Here, we demonstrated that both wild-type and a mutant gamma chain lacking the CH1 domain bind BiP when they are reduced in vivo. However, only oxidized mutant H chain dimers are released from BiP interaction, whereas oxidized wild-type gamma chain dimers still bind BiP. In light (L) chain-producing cells, some of the mutant H chains accumulate with L chains in ER-derived vesicles and some are secreted as IgG. Furthermore, only half of the secreted antibodies bind antigen. We found the same with a mutant gamma chain, in which the CH1 domain was replaced by a CH3 domain. Therefore, we propose that BiP interaction with incompletely folded CH1 domains is required to mediate correct assembly of H and L chains.

In the absence of immunoglobulin heavy- chain expression, some immunoglobulin light (L) chains are retained and degraded within the cell. We investigated the fate of two different nonsecreted murine L chains which exhibit different half-lives (50 min and 3-4 hr). Our results demon- strate that both nonsecreted L chains are quantitatively bound to BiP as partially oxidized molecules. The kinetics of L-chain degradation coincided with those of L-chain dissociation from BiP, which suggests that these two processes are functionally related. L-chain degradation does not depend on vesicular transport, indicating that these soluble proteins are degraded in the endoplasmic reticulum (ER). In contrast, secreted L chains, which interact only transiently with BiP, are com- pletely oxidized and are not degraded even when they are artificially retained in the ER. Our data support the model that, by means of BiP interaction, the ER degradation mech- anism has the potential to discriminate between partially and completely folded molecules.

IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.

BiP is a constitutively-expressed resident protein of the endoplasmic reticulum (ER) of all eucaryotic cells, and belongs to the highly conserved hsp70 protein family. In the ER, BiP is involved in polypeptide translocation, protein folding and presumably protein degradation as well. These functions are essential to cell viability, as has been shown for yeast. In this review, I will summarize the structural features of hsp70 proteins and focus on those experiments which revealed the biological function of BiP.

A cDNA fragment coding for the sex-inducing glycoprotein of Volvox carteri f. nagariensis was expressed in a mammalian cell system (baby hamster kidney (BHK) cells). The transfection product exhibited a specific biological activity intermediate between the natural pheromone of the strains Volvox carteri f. nagariensis and Volvox carteri f. weismannia. Immunoblot analysis showed that the sex-inducing activity was expressed as a set of three iso-glycoproteins (35, 34 and 31 kDa).

Here we show that not only transport defective but all immunoglobulin light chains interact with BiP. Association of BiP with its ligand takes place during or shortly after translation of the light chains. The biological half life of the BiP-light chain complex depends on the fate of the light chains. Light chains which are secreted interact with BiP for only a very short time. In contrast, the complex is biologically more stable in cells which do not secrete their L chains. In these cells, dissociation from BiP correlates with the biological half life of the L chains arguing for a degradation pathway in the endoplasmic reticulum. Instead of being degraded in association with its ligand, BiP is released from the complex and binds to newly synthesized polypeptides. These results support the notion that both H and L chains require the chaperoning function of BiP before or during the process of antibody assembly.