Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis.

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4-7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.

The oligomeric state of Derlin-1 is modulated by endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is a major site of protein synthesis in eukaryotes. Newly synthesized proteins are monitored by a process of quality control, which removes misfolded or unassembled polypeptides from the ER for degradation by the proteasome. This requires the retrotranslocation of the misfolded proteins from the ER lumen into the cytosol via a pathway that, for some substrates, involves members of the recently discovered Derlin family. The Derlin-1 isoform is present as a dimer in the ER, and we now show that its dimerization is modulated by ER stress. Three distinct types of chemically-induced ER stress substantially reduce the levels of Derlin-1 dimer as assayed by both cross-linking and co-immunoprecipitation. The potential function of the different Derlin-1 populations with respect to ER quality control is investigated by analysing their capacity to associate with a misfolded membrane protein fragment. We show for the first time that Derlin-1 can associate with an aberrant membrane protein fragment in the absence of the viral component US11, and conclude that it is the monomeric form of Derlin-1 that interacts with this potential ER-associated degradation substrate. On the basis of these data we propose a model where the pool of active Derlin-1 in the ER membrane can be modulated in response to ER stress.

Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes.

Autophagy, fundamentally a lysosomal degradation pathway, functions in cells during normal growth and certain pathological conditions, including starvation, to maintain homeostasis. Autophagosomes are formed through a mechanism that is not well understood, despite the identification of many genes required for autophagy. We have studied the mammalian homologue of Atg9p, a multi-spanning transmembrane protein essential in yeast for autophagy, to gain a better understanding of the function of this ubiquitious protein. We show that both the N- and C-termini of mammalian Atg9 (mAtg9) are cytosolic, and predict that mAtg9 spans the membrane six times. We find that mAtg9 is located in the trans-Golgi network and late endosomes and colocalizes with TGN46, the cation-independent mannose-6-phosphate receptor, Rab7 and Rab9. Amino acid starvation or rapamycin treatment, which upregulates autophagy, causes a redistribution of mAtg9 from the TGN to peripheral, endosomal membranes, which are positive for the autophagosomal marker GFP-LC3. siRNA-mediated depletion of the putative mammalian homologue of Atg1p, ULK1, inhibits this starvation-induced redistribution. The redistribution of mAtg9 also requires PI 3-kinase activity, and is reversed after restoration of amino acids. We speculate that starvation-induced autophagy, which requires mAtg9, may rely on an alteration of the steady-state trafficking of mAtg9, in a Atg1-dependent manner.

Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon.

We used a site-specific crosslinking approach to study the membrane integration of the polytopic protein opsin at the endoplasmic reticulum. We show that transmembrane domain 1 occupies two distinct Sec61-based environments during its integration. However, transmembrane domains 2 and 3 exit the Sec61 translocon more rapidly in a process that suggests a displacement model for their integration where the biosynthesis of one transmembrane domain would facilitate the exit of another. In order to investigate this hypothesis further, we studied the integration of the first and third transmembrane domains of opsin in the absence of any additional C-terminal transmembrane domains. In the case of transmembrane domain 1, we found that its lateral exit from the translocon is clearly dependent upon the synthesis of subsequent transmembrane domains. By contrast, the lateral exit of the third transmembrane domain occurred independently of any such requirement. Thus, even within a single polypeptide chain, distinct transmembrane domains display different requirements for their integration through the endoplasmic reticulum translocon, and the displacement of one transmembrane domain by another is not a global requirement for membrane integration.

Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon.

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.

A misassembled transmembrane domain of a polytopic protein associates with signal peptide peptidase.

The endoplasmic reticulum (ER) exerts a quality control over newly synthesized proteins and a variety of components have been implicated in the specific recognition of aberrant or misfolded polypeptides. We have exploited a site-specific cross-linking approach to search for novel ER components that may specifically recognize the misassembled transmembrane domains present in truncated polytopic proteins. We find that a single probe located in the transmembrane domain of a truncated opsin fragment is cross-linked to several ER proteins. These components are distinct from subunits of the Sec61 complex and represent a 'post-translocon' environment. In this study, we identify one of these post-translocon cross-linking partners as the signal peptide peptidase (SPP). We find that the interaction of truncated opsin chains with SPP is mediated by its second transmembrane domain, and propose that this interaction may contribute to the recognition of misassembled transmembrane domains during membrane protein quality control at the ER.

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein.

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.