Signal recognition particle receptor-ß (SR-ß) coordinates cotranslational N-glycosylation.

Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the [Formula: see text] subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation-competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR-[Formula: see text] guanosine triphosphate binding site cause an N-glycosylation-deficient phenotype. Neither method alters the association of SR-[Formula: see text] with SR-[Formula: see text], but both approaches reduce the association of SR-[Formula: see text] with the oligosaccharyltransferase complex. These experiments demonstrate that SR-[Formula: see text] has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

Lack of NKG2D in MAGT1-deficient patients is caused by hypoglycosylation.

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins.

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae.

Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal-to-C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y-derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.

Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites.

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Selective inhibition of N-linked glycosylation impairs receptor tyrosine kinase processing.

Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase - STT3A and STT3B - to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases - the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) - at the cell surface in STT3A-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in STT3A-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation.

Mutations in MAGT1 lead to a glycosylation disorder with a variable phenotype.

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.

Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.

Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpesvirus responsible for oral lesions and herpesviral encephalitis. The HSV-1 envelope contains N-glycosylated proteins involved in infection and that are candidate drug targets. NGI-1 is a small-molecule inhibitor of oligosaccharyltransferase (OST) complexes STT3A-OST and STT3B-OST, which catalyze cotranslational and post-translational N-glycosylation, respectively. Because host OSTs attach HSV-1 glycans, NGI-1 might have anti-HSV-1 activity. We evaluated HSV-1 function using NGI-1 and human embryonic kidney 293 knockout lines for OST isoform-specific catalytic and accessory subunits. N-glycosylation of 2 representative envelope proteins (gC and gD) was primarily dependent upon STT3A-OST, but to a large extent replaceable by STT3B-OST. Knockouts impairing STT3A- or STT3B-OST activity, by themselves, did not appreciably affect HSV-1 function (plaque-forming units, normalized to viral particles measured by unglycosylated capsid protein VP5 content). However, with cells lacking STT3B-OST activity (missing the catalytic subunit STT3B or the oxidoreductase subunits magnesium transporter 1/tumor suppressor candidate 3) and thus solely dependent upon STT3A-OST for N-glycosylation, NGI-1 treatment resulted in HSV-1 having cell type-dependent dysfunction (affecting infectivity with Vero cells much more than with the 293 lines). Ablation of post-translational N-glycosylation can therefore make HSV-1 infectivity, and possibly masking of immunogenic peptide epitopes by glycans, highly sensitive to pharmacological inhibition of cotranslational N-glycosylation.-Lu, H., Cherepanova, N. A., Gilmore, R., Contessa, J. N., Lehrman, M. A. Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.

Factor VIII and vWF deficiency in STT3A-CDG.

STT3A-CDG (OMIM# 615596) is an autosomal recessive N-linked glycosylation disorder characterized by seizures, developmental delay, intellectual disability, and a type I carbohydrate deficient transferrin pattern. All previously reported cases (n = 6) have been attributed to a homozygous pathogenic missense variant c.1877C>T (p.Val626Ala) in STT3A. We describe a patient with a novel homozygous likely pathogenic missense variant c.1079A>C (p.Tyr360Ser) who presents with chronically low Factor VIII (FVIII) and von Willebrand Factor (vWF) levels and activities in addition to the previously reported symptoms of developmental delay and seizures. VWF in our patient's plasma is present in a mildly hypoglycosylated form. FVIII antigen levels were too low to quantify in our patient. Functional studies with STT3A-/- HEK293 cells showed severely reduced FVIII antigen and activity levels in conditioned media <10% expected, but normal intracellular levels. We also show decreased glycosylation of STT3A-specific acceptors in fibroblasts from our patient, providing a mechanistic explanation for how STT3A deficiency leads to a severe defect in FVIII secretion. Our results suggest that certain STT3A-dependent N-glycans are required for efficient FVIII secretion, and the decreased FVIII level in our patient is a combined effect of both severely impaired FVIII secretion and lower plasma VWF level. Our report expands both the genotype and phenotype of STT3A-CDG; demonstrating, as in most types of CDG, that there are multiple disease-causing variants in STT3A.

Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.

Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.

Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis.

Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc3Man9GlcNAc2-LLO but not Man9GlcNAc2-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST-dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST-dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors.

The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors. Using this approach, pharmacophores that increase STT3B selectivity are characterized and an STT3B-specific inhibitor is identified. This inhibitor has discrete biological effects on endogenous STT3B target proteins such as COX2 but does not activate the cellular unfolded protein response. Together this work demonstrates that subsets of glycoproteins can be regulated through pharmacologic inhibition of N-linked glycosylation.

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations.

The Sec61 complex is the primary cotranslational protein translocation channel in yeast (Saccharomyces cerevisiae). The structural transition between the closed inactive conformation of the Sec61 complex and its open and active conformation is thought to be promoted by binding of the ribosome nascent-chain complex to the cytoplasmic surface of the Sec61 complex. Here, we have analyzed new yeast Sec61 mutants that selectively interfere with cotranslational translocation across the endoplasmic reticulum. We found that a single substitution at the junction between transmembrane segment TM7 and the L6/7 loop interferes with cotranslational translocation by uncoupling ribosome binding to the L6/7 loop from the separation of the lateral gate transmembrane spans. Substitutions replacing basic residues with acidic residues in the C-terminal tail of Sec61 had an unanticipated impact upon binding of ribosomes to the Sec61 complex. We found that similar charge-reversal mutations in the N-terminal tail and in cytoplasmic loop L2/3 did not alter ribosome binding but interfered with translocation channel gating. These findings indicated that these segments are important for the structural transition between the inactive and active conformations of the Sec61 complex. In summary our results have identified additional cytosolic segments of the Sec61 complex important for promoting the structural transition between the closed and open conformations of the complex. We conclude that positively charged residues in multiple cytosolic segments, as well as bulky hydrophobic residues in the L6/7-TM7 junction, are required for cotranslational translocation or integration of membrane proteins by the Sec61 complex.

Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.

Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII.

N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N-glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N-glycans.

DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon.

In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an STT3A-/- phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2- and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes.

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex.

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.