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Sec61 Complex Subcellular Localisation Journal of Cell Science 112, 1477-1486 (1999) 1477 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0213 The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment Julia J. A. Greenfield and Stephen High* School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK *Author for correspondence (e-mail: [email protected]) Accepted 12 February; published on WWW 22 April 1999 SUMMARY The heteromeric Sec61 complex is composed of α, β and γ in both the ER and the ERGIC/Golgi fractions of the subunits and forms the core of the mammalian ER gradient. The presence of the Sec61 subunits in a post-ER translocon. Oligomers of the Sec61 complex form a compartment suggests that these proteins can escape the transmembrane channel where proteins are translocated ER and be recycled back, despite the fact that none of them across and integrated into the ER membrane. We have contain any known membrane protein retrieval signals studied the subcellular localisation of the Sec61 complex such as cytosolic di-lysine or di-arginine motifs. We also using both wild-type COS1 cells and cells transfected found that another translocon component, the glycoprotein with GFP-tagged Sec61α. By double labelling TRAM, was present in post-ER compartments as immunofluorescence microscopy the GFP-tagged Sec61α demonstrated by subcellular fractionation. Our data was found in both the ER and the ER-Golgi intermediate indicate that the core components of the mammalian ER compartment (ERGIC) but not in the trans-Golgi network. translocon are not permanently resident in the ER, but Immunofluorescence studies of endogenous Sec61β and rather that they are maintained in the ER by a specific Sec61γ showed that these proteins are also located in both retrieval mechanism. the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Key words: Endoplasmic reticulum, Translocation, GFP, ERGIC, ER Sec61α, β and γ, and GFP-tagged Sec61α, are all present retrieval INTRODUCTION known ER retention or retrieval signals normally associated with resident ER proteins. ER resident proteins are generally The Sec61 complex is a core component of the mammalian believed to be retained in the ER either by their exclusion from translocon, forming a channel across the endoplasmic transport vesicles that exit the ER, or by their specific retrieval reticulum (ER) membrane through which newly synthesised from post ER compartments, or by a combination of both secretory proteins are translocated and via which membrane mechanisms (Harter and Wieland, 1996). proteins are integrated (High and Laird, 1997; Rapoport et al., Proteins that have been shown to be exclusively located in 1996). In addition to its central role in protein translocation and the ER, and that do not therefore apparently require any integration at the ER membrane, the Sec61 complex has also specific retrieval mechanism, include cytochrome p450 been shown to be capable of the retrograde transport, or (Szczesna-Skorupa and Kemper, 1993) and aldehyde dislocation, of misfolded proteins from the ER and into the dehydrogenase (Masaki et al., 1996). In the case of cytochrome cytosol prior to their degradation via the proteosome (Wiertz p450, it has been suggested that the large, bulky, cytoplasmic et al., 1996). domain may be responsible for its exclusion from transport The Sec61 complex is composed of three subunits, α, β and vesicles; and indeed ER retention information has been γ, which form a heterotrimer (Rapoport et al., 1996) and localised both in this region and in the N-terminal multiple copies of these heterotrimers surround a central transmembrane domain (Szczesna-Skorupa et al., 1995). It has aqueous pore which spans the ER membrane (Beckmann et al., also been proposed that some ER proteins, such as the 1997; Hanein et al., 1996). Whilst Sec61α is a polytopic ribophorins, form an interacting network of supramolecular protein with ten transmembrane spans, Sec61β and Sec61γ complexes that are excluded from transport vesicles (Ivessa et span the membrane only once (Rapoport et al., 1996). al., 1992). Although the Sec61 complex is known to play a crucial role in Specific motifs that mediate ER residency by retention have promoting translocation at the ER membrane, its exact not yet been described; in contrast, all retrieval mechanisms subcellular location has not been studied in any detail. appear to involve specific signals within the sequences of the Furthermore, none of the Sec61 subunits contain any of the resident ER proteins. The best characterised retrieval signal is 1478 J. J. A. Greenfield and S. High the KDEL sequence at the C terminus of ER lumenal proteins resident proteins that have escaped the ER (Bannykh et al., of mammalian cells (Pelham, 1990). Proteins with a KDEL 1998). In other systems, particularly the yeast Saccharomyces retrieval motif bind to the KDEL receptor (ERD2) and are then cerevisiae, it has been suggested that certain ER proteins, for recycled back to the ER (Lewis and Pelham, 1992a). The example some ER chaperones and components of the KDEL receptor is mainly localised in the first post-ER membrane translocation machinery, are efficiently sorted away compartment, the ERGIC (ER to Golgi intermediate from cargo destined for export at the ER. These components compartment) together with the cis-Golgi (Griffiths et al., are therefore largely excluded from the transport vesicles 1994). As a consequence of its retrieval function, the KDEL leaving the ER (reviewed by Aridor and Balch, 1996). receptor has also been localised to the ER, albeit to a lesser We have studied the localisation of the Sec61 complex in extent than to the ERGIC or cis-Golgi (Griffiths et al., 1994). mammalian cells using two approaches, namely fluorescence A number of single-spanning membrane proteins have a microscopy and subcellular fractionation. The wild-type carboxy-terminal di-lysine or an amino-terminal di-arginine Sec61α protein was tagged with the green fluorescent protein retrieval motif (reviewed by Teasdale and Jackson, 1996), and (GFP) in order to facilitate its visualisation in both fixed and these are found in type I and type II membrane proteins, live cells, and to establish its subcellular location. The respectively. COP1 subunits have been shown to bind directly subcellular location of the endogenous Sec61β and Sec61γ to proteins bearing the cytosolic di-lysine motif and are subunits were also studied by indirect immunofluorescence believed to function in the retrieval of the proteins to the ER microscopy, and in all cases double labelling studies were (Cosson and Letourneur, 1994). Very little is known about the carried out. Taken together with the results from the subcellular ER retention and retrieval of multiple spanning membrane fractionation studies we find that a proportion of all three proteins. subunits of the Sec61 complex are found beyond the ER of In order to ensure the minimum leakage of proteins from the mammalian cells and are clearly present in the ERGIC. We ER some proteins possess a combination of both retention and suggest that the Sec61 complex is transported between the ER retrieval signals. Hence, Sec12p, a yeast protein involved in the and the ERGIC, and that its ER location is dependent upon a formation of ER transport vesicles, has a transmembrane recycling mechanism. domain that acts as a retention signal and an N-terminal cytoplasmic domain which is involved in retrieval (Sato et al., 1996). Tang et al. (1997) later showed that the transmembrane MATERIALS AND METHODS domain was responsible for the formation of oligomers, and that this was the molecular basis for preventing the exit of Materials Sec12p from the ER. Clearly proteins which lack any COS1 African green monkey kidney SV40 transformed cell line retention/retrieval information in their sequence can be (ECACC no. 88031701) was obtained from the European collection retained in the ER if they are in a stable complex with other of animal cell cultures. proteins that do contain such information (Zhen et al., 1995). The Sec61α canine cDNA was a gift from T. Rapoport (Harvard The ER-Golgi intermediate compartment, or ERGIC, (also Medical School, Boston, USA). Restriction enzymes used for known as the intermediate compartment or ‘vesicular tubular recombinant DNA procedures were obtained from New England structures’), is the first recycling compartment of the Biolabs (Hitchin, UK) and Promega (Southampton, UK). T4 DNA ligase and alkaline phosphatase were from Boehringer Mannheim mammalian secretory pathway. It was originally identified as (Lewes, UK). PCR was carried out using QuikChangeTM site-directed the compartment where Semliki Forest virus-infected cells mutagenesis kit (Stratagene, Cambridge, UK). 35[S]methionine was accumulated envelope glycoproteins when intracellular from NEN-Dupont (Stevenage, UK). Bismaleimidohexane (BMH) transport was arrested by incubation at 15°C (Saraste and cross-linking reagent was from Pierce and Warriner (Chester,UK). Kuismanen, 1984). It is usually defined by the presence of Rabbit reticulocyte lysate and the amino acid mixture minus resident marker proteins such as human ERGIC53 (Schweizer methionine were from Promega. The 7-methylguanosine 5′ et al., 1988), its rat homologue p58 (Saraste and Svensson, monophosphate was from Ambion (Austin Texas). Protein A and G 1991) and the KDEL receptor (Tang et al., 1993). These Sepharose came from Zymed (San Francisco,
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