N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

The cotranslational maturation program for the type II membrane glycoprotein influenza neuraminidase

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N- and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regio-specific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.     

Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.     

The use of calnexin and calreticulin by cellular and viral glycoproteins

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. We report here that most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associated more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells.     

Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins.

The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Calnexin, calreticulin, and ERp57

In eukaryotic cells, the endoplasmic reticulum (ER) plays an essential role in the synthesis and maturation of a variety of important secretory and membrane proteins. For glycoproteins, the ER possesses a dedicated maturation system, which assists folding and ensures the quality of final products before ER release. Essential components of this system include the lectin chaperones calnexin (CNX) and calreticulin (CRT) and their associated co-chaperone ERp57, a glycoprotein specific thiol-disulfide oxidoreductase. The significance of this system is underscored by the fact that CNX and CRT interact with practically all glycoproteins investigated to date, and by the debilitating phenotypes revealed in knockout mice deficient in either gene. Compared to other important chaperone systems, such as the Hsp70s, Hsp90s and GroEL/GroES, the principles whereby this system works at the molecular level are relatively poorly understood. However, recent structural and biochemical data have provided important new insights into this chaperone system and present a solid basis for further mechanistic studies.     

Protein folding and maturation in a cell-free system.

Reduced cellular systems have provided important tools to study complex cellular processes. Here we describe the oxidation, oligomerization, and chaperone binding of the viral glycoprotein influenza hemagglutinin in a cell-free system. The cell-free system, comprised of rough endoplasmic reticulum derived microsomes and a reticulocyte lysate, supported the complete maturation of hemagglutinin from the earliest oxidative intermediate to the mature homo-oligomer. Hemagglutinin disulfide bond formation and oligomerization were found to occur in a time- and temperature-dependent manner. Hemagglutinin's temporal association with the molecular chaperones calnexin and calreticulin was similar to that observed for their association with elongating ribosome-attached nascent chains in live cells. Furthermore, a procedure is described that permits the translocation of protein into microsomes that are depleted of lumenal contents. This cell-free system, therefore, provided an effective means to study the biological maturation processes of a protein that traverses the secretory pathway.     

Tyrosinase maturation and oligomerization in the endoplasmic reticulum require a melanocyte-specific factor

Tyrosinase is a glycoprotein responsible for the synthesis of melanin in melanocytes. A large number of mutations have been identified in tyrosinase, with many leading to its misfolding, endoplasmic reticulum (ER) retention, and degradation. Here we describe the folding and maturation of human tyrosinase (TYR) using an in vitro translation system coupled with ER-derived microsomes or with semipermeabilized cells, as an intact ER source. TYR remained misfolded as determined by its sensitivity to trypsin digestion and its persistent interaction with the ER resident lectin chaperones calnexin and calreticulin when produced in ER-derived microsomes or nonmelanocytic semipermeabilized cells. However, when TYR was translocated into semipermeabilized melanocytes, chaperone interactions were transient, maturation progressed to a trypsin-resistant state, and a TYR homodimer was formed. The use of semipermeabilized mouse melanocytes defective for tyrosinase or other melanocyte-specific proteins as the ER source indicated that proper TYR maturation and oligomerization were greatly aided by the presence of wild type tyrosinase and tyrosinase-related protein 1. These findings suggested that oligomerization is a step in proper TYR maturation within the ER that requires melanocyte-specific factors.     

Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.     

Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin

Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.     

Chaperones and folding of MHC class I molecules in the endoplasmic reticulum

In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I. MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved. Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours. This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted. Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.     

Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.     

Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.     

Hepatic lipase maturation: a partial proteome of interacting factors

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.     

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.     

A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.     

Structural and functional relationships between the lectin and arm domains of calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.     

BiP and PDI cooperate in the oxidative folding of antibodies in vitro

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.