Cholera toxin is exported from microsomes by the Sec61p complex

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate-dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex-mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.     

Molecular mechanisms of aquaporin biogenesis by the endoplasmic reticulum Sec61 translocon

The past decade has witnessed remarkable advances in our understanding of aquaporin (AQP) structure and function. Much, however, remains to be learned regarding how these unique and vitally important molecules are generated in living cells. A major obstacle in this respect is that AQP biogenesis takes place in a highly specialized and relatively inaccessible environment formed by the ribosome, the Sec61 translocon and the ER membrane. This review will contrast the folding pathways of two AQP family members, AQP1 and AQP4, and attempt to explain how six TM helices can be oriented across and integrated into the ER membrane in the context of current (and somewhat conflicting) translocon models. These studies indicate that AQP biogenesis is intimately linked to translocon function and that the ribosome and translocon form a highly dynamic molecular machine that both interprets and is controlled by specific information encoded within the nascent AQP polypeptide. AQP biogenesis thus has wide ranging implications for mechanisms of translocon function and general membrane protein folding pathways.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.     

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer’s disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1ΔR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1ΔR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.     

An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein

The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("Gp(alpha)F") is stable, a non-glycosylated version of the same protein ("p(alpha)F") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.     

Impaired transport of intrinsically disordered proteins through the Sec61 and SecY translocon; implications for prion diseases

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.     

The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells

Protein translocons of the mammalian endoplasmic reticulum are composed of numerous functional components whose organization during different stages of the transport cycle in vivo remains poorly understood. We have developed generally applicable methods based on fluorescence resonance energy transfer (FRET) to probe the relative proximities of endogenously expressed translocon components in cells. Examination of substrate-engaged translocons revealed oligomeric assemblies of the Sec61 complex that were associated to varying degrees with other essential components including the signal recognition particle receptor TRAM and the TRAP complex. Remarkably, these components not only remained assembled but also had a similar, yet distinguishable, organization both during and after nascent chain translocation. The persistence of preassembled and complete translocons between successive rounds of transport may facilitate highly efficient translocation in vivo despite temporal constraints imposed by ongoing translation and a crowded cellular environment.     

Protein dislocation from the endoplasmic reticulum--pulling out the suspect

Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.     

Jid1 is a J-protein functioning in the mitochondrial matrix, unable to directly participate in endoplasmic reticulum associated protein degradation

J-proteins are a class of molecular chaperones that serve to stimulate the activity of Hsp70s and are often located to recruit Hsp70 to a particular cellular function. Protein degradation associated with the endoplasmic reticulum (ERAD) is one such cellular process that requires Hsp70 on both faces of the endoplasmic reticulum. At least five J-proteins, including Jid1 (DnaJ protein Involved in ER-associated Degradation), have been implicated in controlling ERAD. Here we show that Jid1 is confined within the mitochondrial matrix - Jid1 has the same topology as the J-proteins Pam18 and Mdj2, which stimulate mitochondrial Hsp70 to drive protein import into the mitochondrial matrix. The location of Jid1 within mitochondria makes it unavailable to participate directly in the regulation of ERAD.     

Structure of the mammalian ribosome-channel complex at 17A resolution

The co-translational translocation of proteins into the endoplasmic reticulum (ER) lumen and the biogenesis of membrane proteins require ribosome binding to a membrane channel formed by the Sec61p complex. We now report the 17A structure of a mammalian ribosome-channel complex derived from ER membranes. Atomic models of the ribosomal subunits were aligned to the programmed ribosome from Thermus thermophilus, to provide a common reference frame. The T.thermophilus ribosome, and by extension all known high resolution subunit models, were then docked within our map of the ribosome-channel complex. The structure shows that the ribosome contains a putative tRNA in the exit site, and a comparison with a non-programmed, yeast ribosome suggests that the L1 stalk may function as a gate in the tRNA exit path. We have localized six major expansion segments in the large subunit of the vertebrate ribosome including ES27, and suggest a function for ES30.The large ribosomal subunit is linked to the channel by four connections. We identified regions in the large subunit rRNA and four proteins that may help form the connections. These regions of the ribosome probably serve as a template to guide the assembly of the asymmetric translocation channel. Three of the connections form a picket fence that separates the putative translocation pore from the attachment site of an additional membrane component. The ribosome-channel connections also create an open junction that would allow egress of a nascent chain into the cytosol. At a threshold that is appropriate for the entire complex, the channel is rather solid and the lumenal half of the putative translocation pore is closed. These data suggest that the flow of small molecules across the membrane may be impeded by the channel itself, rather than the ribosome-channel junction.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

BiP-mediated closing of the Sec61 channel limits Ca(2+) leakage from the ER

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.     

DER7, encoding alpha-glucosidase I is essential for degradation of malfolded glycoproteins of the endoplasmic reticulum

Proteins entering the endoplasmic reticulum (ER) have to acquire an export-competent structure before they are delivered to their final destination. This folding process is monitored by an ER protein quality control system. Folding-incompetent conformers are eliminated via a mechanism called ER-associated protein degradation (ERAD). Genetic studies in the yeast Saccharomyces cerevisiae have revealed that carbohydrate modification plays a crucial role in these processes. Here we show that a previously isolated der mutant (der7-1) is defective in ERAD. We identify DER7 as the gene encoding N-glycan-processing alpha-glucosidase I (EC 3.2.1.106) of the ER and demonstrate that its inactivity, due to a substitution of the conserved glycine residue at position 725 by arginine (G725R) in the der7-1 mutant, leads to ER-stress.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.