1958 Research Article Different effects of Sec61a, Sec62 and Sec63 depletion on transport of polypeptides into the of mammalian cells

Sven Lang1, Julia Benedix1, Sorin V. Fedeles2, Stefan Schorr1, Claudia Schirra3, Nico Scha¨uble1, Carolin Jalal1, Markus Greiner1, Sarah Haßdenteufel1,Jo¨ rg Tatzelt4, Birgit Kreutzer5, Ludwig Edelmann6, Elmar Krause3, Jens Rettig3, Stefan Somlo2, Richard Zimmermann1,* and Johanna Dudek1 1Medical Biochemistry and Molecular Biology, Saarland University D-66421, Homburg, Germany 2Internal Medicine, Yale School of Medicine, New Haven, CT 06520-8029, USA 3Institute of Physiology, Saarland University, D-66421, Homburg, Germany 4Neurobiochemistry, Adolf-Butenandt-Institute, Ludwig-Maximilians-University and German Center for Neurodegenerative Diseases, D-80336 Munich, Germany 5Clinic of Urology and Pediatric Urology, Saarland University Hospital, D-66421 Homburg, Germany 6Institute of Anatomy and Cell Biology, Saarland University, D-66421 Homburg, Germany *Author for correspondence ([email protected])

Accepted 12 December 2011 Journal of Cell Science 125, 1958–1969 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.096727

Summary Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 in human cells affects co- and post-translational transport of presecretory into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored . Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Key words: ER protein transport, Sec61 complex gating, Sec62 protein, Sec63 protein, Tail-anchored membrane proteins Journal of Cell Science

Introduction dispensable for post-translational precursor protein targeting that Protein transport into the endoplasmic reticulum (ER) is the first has been observed for small presecretory proteins (Schlenstedt and step in the biogenesis of most secreted proteins and many Zimmermann, 1987; Mu¨ller and Zimmermann, 1987; Mu¨ller organellar proteins in mammalian cells (Zimmermann et al., and Zimmermann, 1988; Schlenstedt et al., 1990). This post- 2011). Furthermore, integration into the ER membrane is the first translational transport is typically facilitated by cytosolic step in the biogenesis of many eukaryotic membrane proteins molecular chaperones, such as Hsc70 and Hsp40 (Dierks et al., (Borgese and Fasana, 2011). Typically, protein transport into the 1993). Initial co- and post-translational membrane insertion and ER involves N-terminal signal peptides or C-terminal tail anchors completion of translocation involve the Sec61 complex, in the precursor proteins and occurs co- or post-translationally. In comprising a-, b-andc-subunits (Go¨rlich et al., 1992; Hartmann the case of signal-peptide-containing precursors, transport and et al., 1994; Wirth et al., 2003; Van den Berg et al., 2004; Becker membrane integration can be divided into three stages: targeting, et al., 2009). However, these mechanisms have never been directly initial membrane insertion (at the Sec61 complex, see below) demonstrated at the cellular level. Following initial membrane and completion of membrane integration or translocation. Co- insertion, both types of precursor protein are typically processed by translational precursor protein targeting involves cleavable or non- signal peptidase and/or oligosaccharyl transferase. cleavable signal peptides at or near the N-terminus of the precursor In addition to the Sec61 complex, the ER-membrane- polypeptides plus the ribosome, the signal recognition particle integrated Hsp40 chaperone Sec63p and its lumenal Hsp70 (SRP), the SRP receptor (SR) (Walter and Blobel, 1981; Meyer partner Kar2p (also termed BiP) were found to be essential and and Dobberstein, 1980) and the ribosome receptor that is identical to be involved in co- and post-translational transport in to the Sec61 complex (Kalies et al., 1994). Alternatively, synthesis Saccharomyces cerevisiae (Brodsky et al., 1995; Young et al., of the respective precursor proteins can be initiated at ribosomes 2001). However, the function of mammalian Sec63 protein that are permanently associated with the ER (Potter et al., 2001), remains elusive, even though Sec63 is known to occur in thus eliminating the involvement of the SRP and SR. The SRP, SR stoichiometric amounts in canine pancreatic microsomes relative and ribosomes but not the N-terminal signal peptides are to the a-subunit of the Sec61 complex, and in association with Protein transport into the mammalian ER 1959

this complex (Tyedmers et al., 2000). Mammalian BiP (also of various TA proteins. In addition, we addressed the putative known as 78 kDa glucose-regulated protein; Grp78) appears to roles of mammalian Sec62 and Sec63 in protein transport into the have two functions in protein translocation, it is involved in the ER with the same experimental strategy. The silencing of the insertion of precursor polypeptides into the Sec61 complex SEC63 gene only affected a subset of precursor proteins. This (assayed as processing by signal peptidase) or opening of the phenotype was reproduced in murine Sec632/2 cells. By contrast, Sec61 channel (Klappa et al., 1991; Dierks et al., 1996), and it silencing of the SEC62 gene inhibited post-translational transport binds to the incoming precursor polypeptide and acts as a of a -containing precursor protein. molecular ratchet, thereby facilitating completion of translocation (assayed as sequestration) (Nicchitta and Blobel, 1993; Tyedmers Results et al., 2003; Shaffer et al., 2005). Analogous to the situation in The human SEC61A1 gene is essential for cell growth Saccharomyces cerevisiae (Brodsky et al., 1995; Young et al., and viability 2001), Sec63 could recruit BiP to the Sec61 complex and to In order to demonstrate that the Sec61 complex is involved in translating ribosomes; Sec63 also activates BiP for interaction protein transport into the ER in mammalian cells, the effect of with its substrates (Tyedmers et al., 2000). However, Go¨rlich and silencing of the SEC61A1 gene had to be analyzed. HeLa cells Rapoport reported that Sec63 and BiP are not required for were treated twice with candidate siRNAs for various incubation reconstitution of protein translocation using purified membrane times. Western blot analysis showed that two different siRNAs proteins from the mammalian ER and model precursor that were directed against the coding (SEC61A1) and untranslated polypeptides such as preprolactin, preimmunoglobulin k light (SEC61A1-UTR) regions of the SEC61A1 gene were the most chain and VSV G protein (Go¨rlich and Rapoport, 1993). efficient at silencing the gene when used individually at a The ER membrane protein Sec62 is associated with the Sec61 concentration of 20 nM or used in combination at 10 nM each complex and Sec63 (Meyer et al., 2000; Tyedmers et al., 2000) as (supplementary material Fig. S1A). Whether used alone or in well as with ribosomes on the ER surface in mammalian cells combination, there was maximum silencing (,90%) after 96 (Mu¨ller et al., 2010). The yeast ortholog Sec62p is only involved hours of incubation. At that time, the growth and viability of the in post-translational protein transport into the yeast ER and silenced cells only began to be affected compared with cells supposedly forms a signal peptide receptor together with the that had been transfected with a negative control siRNA proteins Sec71p and Sec72p (Lyman and Schekman, 1997; Plath (supplementary material Fig. S1B,C; Fig. S2). Between 96 and et al., 1998). Mammalian cells appear to lack orthologs of the 120 hours, however, the SEC61A1-silenced cells started to die. latter two yeast proteins, whereas trypanosomes lack Sec62p As expected, the SEC61A1 gene is essential in human cells. and Sec72p orthologs but contain an ortholog of Sec71p However, these results defined an experimental window between (Zimmermann and Blatch, 2009). The function of mammalian 90 and 100 hours after first transfection with SEC61A1-targeting Sec62 remained elusive, too. siRNAs that allows the functional analysis of the SEC61A1 gene Tail-anchored (TA) membrane proteins use C-terminal tail product. anchors for post-translational integration into the mammalian ER The cells were also characterized by quantitative RT-PCR membrane plus one of a number of different cytosolic factors, (qPCR) at various time points to determine the levels of

Journal of Cell Science such as the SRP, cytosolic molecular chaperones (Hsc70 particular mRNAs (supplementary material Fig. S1D). The and Hsp40) or the TA-dedicated machinery, the so-called qPCR data demonstrated efficient depletion of SEC61A1 transmembrane recognition complex (TRC) (Abell et al., 2004; mRNA at 48 hours. According to the western blot analysis, Abell et al., 2007; Stefanovic and Hegde, 2007; Rabu et al., however, most efficient depletion of the Sec61a1 protein took an 2008). At the level of the ER membrane, certain TA proteins, additional 48 hours (supplementary material Fig. S1A). Silencing such as cytochrome b5 (cytb5), can enter the ER membrane the SEC61A1 gene also affected the levels of the tail-anchored b- unassisted (Brambillasca et al., 2005; Brambillasca et al., 2006). and c-subunits of the Sec61 complex (supplementary material Other TA proteins, however, require the ER-membrane-resident Table S1). At the mRNA level, however, there was no decrease in SR or TRC receptors (Abell et al., 2004; Vilardi et al., 2011). An the SEC61B gene transcription (supplementary material Fig. additional involvement of the Sec61 complex seemed to be a S1D). These results were consistent with a scenario in which possibility for TA proteins, such as synaptobrevin2 (Syb2) and there is degradation of the b- and c-subunits in the absence of the Sec61b, that can involve the SRP and SR (Abell et al., 2004). crucial a-subunit. However, other in vitro experiments have suggested that the The levels of all other tested ER membrane and lumenal Sec61 complex is not involved in membrane integration of TA proteins were not reduced in SEC61A1-silenced cells compared proteins (Kutay et al., 1995; Stefanovic and Hegde, 2007). with control-siRNA-treated cells after 96 hours of SEC61A1 gene Physiologically, TA proteins are not modified by signal peptidase silencing (supplementary material Table S1), suggesting that this or oligosaccharyl transferase. However, the established model silencing time did not result in global secondary effects on the ER TA proteins typically contain glycosylation sites at the C- proteome. Furthermore, BiP and Grp170 mRNA levels as well as terminus in order to allow quantitative analysis of membrane protein levels did not indicate protein misfolding in the ER, i.e. integration. did not show that the unfolded protein response had been We addressed the central role of the human Sec61 complex in activated (supplementary material Fig. S1D; Table S1). However, protein transport into the ER. The strategy was to silence the we detected an upregulation of the SRA and SRB SEC61A1 gene in human cells by employing different SEC61A1- as a result of silencing the SEC61A1 gene (supplementary targeting siRNAs and to study the transport of various precursor material Table S1). Furthermore, the cellular ribosome content proteins into the ER of the respective semi-permeabilized cells. was not affected by SEC61A1 silencing, suggesting that no Although silencing of the SEC61A1 gene affected signal-peptide- adaptive processes in global protein synthesis had been initiated dependent protein transport in general, it did not affect transport (supplementary material Table S1). 1960 Journal of Cell Science 125 (8)

Table 1. Transport efficiency of cellular ER after 96 hours silencing with the indicated siRNAs Precursor protein SP TA SEC61 siRNA SEC61-UTR siRNA SEC61 siRNAs Co-translational transport ppl + – 763 (6) 462 (10) 662 (10) ppaf+–6165 (4) 55611 (7) 4867 (7) pERj3 + – 1063 (4) 461 (4) 462 (4) pAQP2* + – 4161 (2) 3464 (2) 3965 (2) pivc* + – 2864 (5) 2766 (5) 1865 (5) Post-translational transport ppcecA + – 2765 (6) 2163 (6) 2666 (6) cytb5-ops28* – + 9666 (9) 9966 (8) 9665 (9) Sec61b-ops13* – + 9862 (7) 9962 (7) 10062 (6) Syb2-ops13* – + 10663 (8) 10264 (8) 11764 (8)

Values are means 6 s.e.m. (n). The processing efficiency (signal peptide cleavage for ppl, ppaf, ppcecA and glycosylation for pERj3, pAQP2, pivc, cytb5- ops28, Sec61b-ops13, Syb2-ops13) of the respective control cells was set to 100%. SP, N-terminal signal peptide; TA, C-terminal tail anchor; *, membrane protein. We note that the translation efficiency for ppl, ppcecA and pivc was at least four times higher than ppaf and pAQP2, which might, together with the strength of the signal peptide, explain the higher or lower sensitivity towards SEC61A1 silencing.

Next, the morphology of the ER in the silenced cells was SEC61A1-targeting siRNAs or the negative control siRNA for 96 characterized by three-dimensional (3D)-structured illumination hours and then semi-permeabilized with digitonin. First, ppl microscopy (3D-SIM) and electron microscopy (EM). HeLa was synthesized in reticulocyte lysate in the presence of cells were treated with control, SEC61A1 or SEC61A1-UTR [35S]methionine. The translation reactions were supplemented siRNA up to 96 hours and then, subjected to 3D-SIM and EM. with equivalent amounts of the different semi-permeabilized Cells treated with control or one of the SEC61A1 siRNAs did siRNA-treated cells; after incubation, all samples were subjected not show gross alterations in the typical ER morphology to protease treatment and SDS-PAGE and phosphorimaging regardless of how much Sec61 complex was present analysis. In the presence of cells incubated with the negative (supplementary material Fig. S3). However, qualitative and control siRNA, ppl was cleaved to the mature prolactin (pl) that quantitative EM analysis demonstrated the central role of the was protected against externally added protease by the ER Sec61 complex as an ER membrane resident ribosome receptor membrane. This demonstrated that pl had entered the ER lumen (Kalies et al., 1994), in that ,60% less ER-bound polysomes (Fig. 2A). In the SEC61A1-silenced cells, ppl was processed to pl were observed after 96 hours of SEC61A silencing, i.e. the less efficiently (between 462% and 763% of the control-siRNA- average number of ER-bound ribosomes was reduced from 17 treated cells; Table 1), and, therefore, there was very little of the per mm in control-siRNA-treated cells to 7 per mmincells protease-protected pl. Similar results were obtained for the depleted of Sec61 complex (Fig. 1). precursors of paf and ERj3 (Table 1). The model membrane- and Journal of Cell Science glycoprotein invariant chain was also analyzed in the transport Silencing the human SEC61A1 gene inhibits signal assay (Fig. 2A; Table 1). In the presence of cells incubated with peptide- but not tail-anchor-dependent protein transport the negative control siRNA, the invariant chain was glycosylated into the ER and partially protected against the externally added protease by The protein transport activity of the Sec61 complex in the the ER membrane, demonstrating that it had entered the ER silenced cells was monitored using an established transport assay membrane. In the SEC61A-silenced cells, the invariant chain was that employs in vitro synthesis of different model precursor glycosylated less efficiently (between 1865% and 2864% of the polypeptides in the presence of semi-permeabilized cells (Wilson control siRNA-treated cells), and very little of the invariant chain et al., 1995). In addition to the presecretory protein preprolactin fragment was protected from the protease. Similar results were (ppl; also known as pre-Prl), several types of precursor were obtained for the precursor of AQP2 (Table 1). Thus, silencing of employed in the transport assay to gain a more general picture of the SEC61A1 gene led to a reduced ability to co-translationally ER protein translocation. These precursor proteins included the import presecretory and membrane protein precursors. presecretory yeast protein prepro-a-factor (ppaf), ERj3 (ER In order to further substantiate the conclusion from SEC61A1 lumen; also known as DnaJ homolog subfamily B member 11 in silencing we attempted expression of the SEC61A1 cDNA, human), the invariant chain of the human class II major lacking the UTR of the SEC61A1 gene, in the presence of the histocompatibility complex (ivc; type II plasma membrane SEC61A1–UTR siRNA and observed that it indeed rescued the protein), aquaporin-2 (AQP2; polytopic plasma membrane phenotype of SEC61A1 silencing, i.e. in contrast to the vector protein) and presecretory protein preprocecropin A (ppcecA; control it partially restored the transport activity (Fig. 2B). post-translationally transported). In the case of precursors of Averaging three signal-peptide-containing precursor proteins, soluble proteins, initial insertion of precursors into the Sec61 processing activity was increased by the SEC61A1 cDNA from complex was assayed as modification by signal peptidase and/or 1666% to 62619% of the control-siRNA-treated cells (data not oligosaccharyl transferase because these two enzymes act on shown). Based on western blot analysis, the complementation incoming polypeptides at the lumenal face of the ER membrane. efficiency of the SEC61A1 expression plasmid was ,64% By contrast, completion of translocation was assayed as protease (n517), which could explain the incomplete rescue. Thus the resistance of the mature protein in the absence of detergent (in observed effects of SEC61A1 silencing were indeed due to the sequestration analysis). HeLa cells were incubated with the two SEC61A1 gene. Also, the levels of Sec61b (6065%, n512) and Protein transport into the mammalian ER 1961

Fig. 1. Effect of SEC61A1 gene silencing on ribosome content of the human ER. Transfected HeLa cells were grown on poly- lysine-coated sapphire discs in 6 cm dishes for 96 hours and subjected to freeze substitution prior to the embedding. Ultrathin sections were cut parallel to the cell monolayer, collected on copper grids, stained with uranyl acetate and lead citrate, and analyzed on a Tecnai 12 Journal of Cell Science Biotwin EM. Representative images are shown. The arrowheads point to putative ER cisternae. The ribosome content of the ER in control siRNA and the two SEC61A1 siRNAs was quantified as 16.961.2% (n519 pictures; 82.5 mm membrane), 6.560.5% (n511; 69.5 mm) and 6.560.7% (n514, 55.7 mm) ribosomes/1 mm. Scale bars: 1 mm (upper row), 0.5 mm (middle row) and 0.2 mm (lower row).

SRb (181611%, n515) partially returned to control levels cells, ppcecA was barely processed (between 2163% and 2765% under these complementation conditions, and complementation of the control siRNA-treated cells) and imported. Thus, silencing of restored cell proliferation and viability (supplementary material the SEC61A1 gene also led to a reduced ability of the ER to post- Fig. S4). translationally import small presecretory proteins. Next, ppcecA, a post-translationally transported model Finally, we studied membrane integration of various TA presecretory protein with a cleavable signal peptide, was proteins, such as cytb5-ops28 (Colombo et al., 2009), Sec61b- examined in the transport assay (Fig. 2C; Table 1). The precursor ops13 and Syb2-ops13 (Rabu et al., 2008) that employ different protein was synthesized in the absence of membranes and, cytosolic factors. SEC61A1 silencing had no effect on membrane subsequently incubated with siRNA-treated semi-permeabilized integration of these TA membrane proteins, as can be deduced cells as indicated. The samples were then subjected to protease from equally efficient glycosylation of the C-terminus under all treatment, SDS-PAGE and phosphorimaging. As expected, in the conditions (Fig. 2D; Table 1). Thus, silencing of the SEC61A1 presence of cells with intact Sec61 complexes, ppcecA was inserted gene did not lead to a reduced ability to import TA proteins and into the Sec61 complex (i.e. processed to mature procecropin A; had no overall effects on the integrity of the ER membrane, as pcecA) and imported into the ER, where it was protected against can be concluded from the activity of oligosaccharyl transferase externally added protease. By contrast, in the SEC61A1-silenced that depends on a lipid-linked oligosaccharide. 1962 Journal of Cell Science 125 (8)

Silencing the SEC62 gene inhibits post-translational transport of small presecretory proteins into the human ER Having established a powerful experimental system to analyze the role of membrane proteins in protein transport into the mammalian ER, we next employed this system for Sec62. An efficient protocol for silencing of the human SEC62 gene had already been developed (Greiner et al., 2011). Western blot analysis characterized two different siRNAs that were directed against the coding (SEC62) and untranslated (SEC62-UTR) regions of the SEC62 gene as most efficient in silencing the gene when used individually at a concentration of 20 nM (Fig. 3A). The maximum silencing (,90%) was observed after 96 hours of incubation. In contrast to the SEC61A1 gene the SEC62 gene might not be essential in HeLa cells, or possibly a small amount of Sec62 is sufficient for growth of HeLa cells (Greiner et al., 2011). The protein transport activity of the SEC62-silenced cells was monitored using the above described transport assays. HeLa cells were incubated with the two SEC62 siRNAs or the negative control siRNA for 96 hours and treated with digitonin. Then, transport assays were carried out for three types of precursor protein: the co-translationally transported precursor protein ppl, the small presecretory protein ppcecA and the tail-anchored protein cytb5. In contrast to the other two types of protein, ppcecA was less efficiently processed and imported after SEC62 silencing under co- and post-translational assay conditions (Fig. 3B,C; Table 2). Thus, silencing of the SEC62 gene led to a reduced ability for post-translational import of a small presecretory protein, but had no effects on co-translational transport or on post-translational membrane integration of TA proteins (Fig. 3D,E). In order to further substantiate this conclusion we attempted expression of the SEC62 cDNA, lacking the UTR of the SEC62 gene, in the presence of the SEC62-UTR siRNA and observed that it rescued the phenotype of SEC62 silencing, i.e. it partially restored the transport activity (Fig. 3F; Journal of Cell Science Table 2). Based on western blot analysis the overexpression of the SEC62 gene under these conditions was ,600%. Thus the observed effect of SEC62 silencing on ppcecA transport was indeed due to the SEC62 gene.

Silencing the human and murine SEC63/Sec63 inhibits transport of only a subset of signal-peptide- containing precursor proteins into the ER We also employed this experimental system for Sec63. First, an efficient protocol for silencing of the SEC63 gene had to be developed. HeLa and NIH/3T3 cells were treated twice with Fig. 2. Effect of SEC61A1 gene silencing on signal peptide- and tail- candidate siRNAs for various times. Western blot analysis anchor-dependent protein transport into the human ER. Before treatment characterized three different siRNAs that were directed against with digitonin for preparation of semi-permeabilized cells, HeLa cells were treated with the indicated SEC61A1 siRNA or control siRNA for 96 hours, the coding (SEC63/Sec63) and SEC63-UTR regions of the where indicated, for the last 48 hours with SEC61A1 plasmid or control SEC63/Sec63 gene as most efficient at silencing the gene when plasmid. A representative western blot for SEC61A1 silencing is shown in used individually at a concentration of 20 nM (Fig. 4A). The supplementary material Fig. S1A. The indicated precursor polypeptides were maximum silencing (,90%) was observed after 96 hours of imported into the indicated ER fractions under co- (A,B) or post-translational incubation. The growth and viability for up to 168 hours (C,D) transport conditions, or incubated in the presence of buffer as a of SEC63/Sec63-silenced cells was indistinguishable from that of negative control. The membranes were subjected to sequestration analysis, control siRNA-treated cells (not shown). Thus, in contrast to the followed by SDS-PAGE and phosphorimaging. Only the areas of interest for SEC61A1 gene the SEC63/Sec63 gene is not essential in human single gels are shown. Processing efficiency was calculated for the mature and murine cells. This notion was further corroborated by the fact proteins in the gel shown (processed and/or glycosylated) as a percentage of the precursor plus mature protein. g, a glycosylated polypeptide; p, precursor; that there was no difference in cell growth and viability between +/+ 2/2 PK, proteinase K; Note that reticulocyte lysate contains residual amounts of Sec63 and Sec63 cells (see below). We note, however, that ER membrane that can give rise to minute amounts of mature proteins in the knockout of the Sec63 gene in mice led to early embryonic absence of added membranes. lethality (Fedeles et al., 2011). Protein transport into the mammalian ER 1963

Fig. 3. Effect of SEC62 gene silencing on signal peptide- and tail-anchor-dependent protein transport into the ER. Before treatment with digitonin for preparation of semi-permeabilized cells, HeLa cells were treated with the indicated SEC62 siRNA or control siRNA for 96 hours (A–F), where indicated; TX100, Triton X-100, for the last 48 hours with SEC62 plasmid or control plasmid (F). (A) A representative western blot documenting SEC62 silencing. The indicated precursor polypeptides were imported into the indicated ER fractions under co- (C,E) or post-translational (B,D,F) transport conditions, or incubated in the presence of buffer as a negative control. The membranes were subjected to sequestration analysis, followed by SDS-PAGE and phosphorimaging. Only the areas of interest for a single gel are shown. Processing efficiency was calculated for the gel shown as described in the legend to Fig. 2. g, glycosylated polypeptide; p, precursor; PK, proteinase K; TX100, Triton X-100. Journal of Cell Science The protein transport activity of the Sec63 in the silenced cells or with either of the two SEC63/Sec63 siRNAs, ppl was cleaved was monitored using the above established transport assay. HeLa to the mature pl (8766% to 102616% of the control siRNA- or NIH/3T3 cells were incubated with the SEC63/Sec63 siRNAs treated cells) that was protected against externally added protease or the negative control siRNA for 96 hours and then treated with by the ER membrane (Table 3). Similar results were obtained for digitonin for permeabilization. Equivalent amounts of cells the precursor of immunoglobulin k (Table 3). By contrast, in the were used for the protein transport activity assay. First, ppl presence of cells incubated with the SEC63/Sec63 siRNAs, the was synthesized in reticulocyte lysate in the presence of precursor of the ER lumenal protein ERj3 was less efficiently [35S]methionine. The translation reactions were supplemented processed and glycosylated to gERj3 (between 4866 and with the different semi-permeabilized siRNA-treated cells; after 56610% of the control-siRNA-treated cells) that was protected incubation, all samples were subjected to protease treatment and against externally added protease by the ER membrane (Fig. 4B; SDS-PAGE and phosphorimaging analysis. In the presence of Table 3). Thus, transport of pERj3 but not of ppl and human or murine cells incubated with the negative control siRNA preimmunoglobulin k into the ER lumen involves Sec63.

Table 2. Transport efficiency of cellular ER after 96 hours silencing with the indicated siRNAs Precursor protein SP TA SEC62 siRNA SEC62-UTR siRNA SEC62-UTR siRNA + SEC62 plasmid Co-translational transport ppl + – 8160 (1) 9762 (2) n.d. ppcecA + – 58610 (2) 7360 (1) n.d. Post-translational transport ppcecA + – 4469 (3) 3867 (6) 9062 (3) cytb5-ops28* – + 9665 (2) 104610 (3) n.d.

Values are means 6 s.e.m. (n). The processing efficiency (signal peptide cleavage for ppl and ppcecA and glycosylation for cytb5-ops28 and Syb2-ops13) of the respective control cells was set to 100%. SP, N-terminal signal peptide; TA, C-terminal tail anchor; n.d., not determined; *, membrane protein. Note that under co-translational transport conditions preprocecropin A is transported in a manner that is SRP and SR dependent as well as independently (Schlenstedt et al., 1990). 1964 Journal of Cell Science 125 (8)

Fig. 4. The absence of Sec63 affects protein transport into the ER in a precursor-specific manner. (A–C) Before treatment with digitonin for preparation of semi-permeabilized cells, HeLa and NIH/3T3 cells were treated with the indicated SEC63/Sec63 siRNA or control siRNA for 96 hours and, where indicated for the last 48 hours with SEC63 plasmid or control plasmid (C). (A) A representative western blot for SEC63 silencing in HeLa cells is shown. The asterisk indicates an unrelated polypeptide in HeLa cells. (D) Murine kidney Sec63+/+ and Sec632/2 cells were treated with digitonin for preparation of semi-permeabilized cells. (B–D) The indicated precursor polypeptides were imported into the cellular ER under co- or post-translational (ppcecA, TA proteins) transport conditions or incubated in the presence of buffer as a negative control. Transport reactions were either followed by sequestration analysis and/or by SDS- PAGE and phosphorimaging. Only the Journal of Cell Science areas of interest from single gels are shown. The efficiencies of modification by signal peptidase or oligosaccharyl transferase are indicated for the mature proteins in the gel shown as a percentage of the precursor plus mature protein. g, glycosylated polypeptide; p, precursor; PK, proteinase K.

Next, the precursor of ERj1 (type I ER membrane protein; also Finally, ppcecA, the model post-translationally transported known as DnaJ C1) was analyzed using the transport assay presecretory protein, was examined in the transport assay (Table 3). In the presence of cells incubated with the negative (Fig. 4B; Table 3). The precursor protein was synthesized in control siRNA or with either one of the SEC63/Sec63 siRNAs, the absence of membranes and subsequently incubated with pERj1 was processed to ERj1 (between 9665% and 10166% of siRNA-treated semi-permeabilized cells as indicated. The the control-siRNA-treated cells). The model membrane proteins samples were then subjected to protease treatment, SDS-PAGE and glycoproteins, ivc and AQP2 were also analyzed in the and phosphorimaging. In the presence of cells with intact Sec63, transport assay (Fig. 4B; Table 3). In the presence of cells ppcecA was inserted into the Sec61 complex (i.e. processed to incubated with the negative control siRNA, both precursors were mature pcecA) and imported. By contrast, in the SEC63/Sec63- glycosylated and partially protected against the externally added silenced cells, ppcecA was less efficiently processed (between protease by the ER membrane, demonstrating that they had 69610% and 7166% of the control-siRNA-treated cells). entered the ER membrane. However, in the SEC63/Sec63- To rule out unspecific effects of SEC63/Sec63 silencing, we silenced cells, the ivc and AQP2 were glycosylated less studied membrane integration of the TA proteins cytb5-ops28 efficiently (between 4060% and 7564% of the control-siRNA- and Syb2-ops13. SEC63/Sec63 silencing had no effect on treated cells), and very little of the ivc and AQP2 was protected membrane integration of these proteins as shown by from the protease. Thus, insertion of AQP2 and invariant chain unimpaired glycosylation in the SEC63/Sec63-silenced cells into the ER membrane, but not ERj1, involves Sec63. (Fig. 4B; Table 3). Thus, silencing of the SEC63/Sec63 gene Protein transport into the mammalian ER 1965

Table 3. Transport efficiencies of cellular ER from HeLa and NIH/3T3 cells after 96 hours silencing with the indicated siRNAs or from murine Sec632/2 cells HeLa HeLa NIH/3T3 Precursor protein SP TA SEC63 siRNA SEC63-UTR siRNA Sec63 siRNA Sec632/2 Co-translational transport ppl + – 8766 (4) 102616 (2) n.d. 8667 (13) pIgk +– 9265 (2) 10465 (4) 94 (1) 10768 (4) pERj3 + – 4866 (4) 56610 (3) 5267 (6) 261 (12) pERj1* + – 9665 (4) 10166 (4) n.d. 101613 (5) pivc* + – 7564 (3) n.d. n.d. 5467 (11) pAQP2* + – 6663 (3) 4060 (1) 62615 (2) 5664 (13) pPrPDHD + – n.d. n.d. n.d. 4067 (4) pPrPDGPI + – n.d. n.d. n.d. 3369 (4) Post-translational transport ppcecA + – 7166 (7) n.d. 69610 (6) 4566 (8) cytb5-ops28* – + 9468 (3) n.d. 10066 (6) 9063 (12) Syb2-ops13* – + 9968 (2) 9362 (2) n.d. 9163 (10)

Values are means 6 s.e.m. (n). The processing efficiency (signal peptide cleavage for ppl, pIgk (pre-immunoglobulin k), pERj1, ppcecA, and glycosylation for pERj3, pAQP2, pivc, pPrP, cytb5-ops28, Syb2-ops13) of the respective control cells was set to 100%. SP, N-terminal signal peptide; TA, C-terminal tail anchor; n.d., not determined; *, membrane protein.

led to a reduced membrane integration or import of certain proteins such as the membrane proteins ERj1, Sec61a, Sec62, precursors, but had no overall effects on the integrity of the SRb and calnexin and the lumenal proteins BiP, Grp170, protein ER membrane, i.e. on glycosylation activity and membrane disulfide-isomerase (PDI) and calreticulin were not reduced in integration or import of other precursor proteins. Sec632/2 cells (supplementary material Table S2). In order to further substantiate this conclusion we also induced a rescue of the SEC63 silencing phenotype in HeLa cells by Sec63 affects transport of prion protein expressing the SEC63 cDNA lacking the SEC63 UTR in the The experiments described above suggested a precursor-specific presence of the SEC63-UTR siRNA (Fig. 4C). The glycosylation role of Sec63 in the early phase of protein transport. The concept efficiency of ERj3 was increased by SEC63 cDNA expression by of substrate-specific gating of the Sec61 channel mediated by a factor of about two (n53) and the complementation efficiency signal sequences was originally put forward for the translocation of the SEC63 expression plasmid was ,9768% (n53). Thus, the of the prion protein (PrP) into the ER (Kim et al., 2002). observed effects of SEC63 silencing were a direct result of a lack Therefore, we included murine PrP in our analysis of the effects of Sec63. of Sec63 depletion. Two variants of prion protein were employed that lack the central hydrophobic region (also termed potential Journal of Cell Science Knockout of the murine Sec63 gene inhibits transport of transmembrane domain; amino acid residues 113–133; DHD) and the same subset of precursor proteins into the ER the GPI acceptor region (residues 231–254, DGPI), respectively In order to address the transport activity of mammalian Sec63 (Winklhofer et al., 2003). Sec632/2 cells and the negative control using an siRNA-independent approach, similar transport Sec63+/+ cells were treated with digitonin. The translation experiments were carried out with murine Sec63 knockout cells reactions were supplemented with the two types of semi- (Fedeles et al., 2011). The protein transport activity of Sec63 was permeabilized cells, and after incubation, all samples were monitored using the above described transport assay. Several subjected to protease treatment and SDS-PAGE and precursor proteins (ppl, pre-immunoglobulin k and pERj1) were phosphorimaging analysis (Fig. 5; Table 3). In the presence of not affected by the absence of Sec63, whereas transport of the Sec632/2 cells the precursors of the two PrP variants were less other precursor proteins (pERj3, pivc, pAQP2, ppcecA) was efficiently glycosylated to gPrP that was protected against affected to varying degrees (Fig. 4D; Table 3). We note that in externally added protease by the ER membrane (average the latter cases, initial insertion into the Sec61 complex was glycosylation: 3668%; n54). Thus, the lack of Sec63 led to a lower in the absence of Sec63, as assayed by signal peptidase defect in transport of PrP into the ER. processing or glycosylation. To control ER integrity in the Sec632/2 cells (including the glycosylation machinery) we Discussion employed the glycosylatable ER TA proteins cytb5 and Syb2 SEC61A1 gene silencing inhibits co- and post-translational (Fig. 4D; Table 3). Both proteins were glycosylated in the transport of presecretory proteins into the ER of deficient cells as efficiently as in control cells. Overall, the mammalian cells but not membrane integration of effects of Sec63 depletion on transport of certain precursor TA proteins proteins were more pronounced in the Sec632/2 cells than in Transport of nascent polypeptides into the membrane or lumen of the SEC63-silenced cells, most probably because of the more the mammalian ER involves the Sec61 complex (Go¨rlich and efficient Sec63 depletion. Rapoport, 1993) that forms both a ribosome receptor (Kalies Quantitative western blotting of Sec63+/+ and Sec632/2 cells et al., 1994) and a gated pore in the ER membrane (Kim et al., confirmed the substrate specificity of Sec63 for transport of 2002; Wirth et al., 2003). So far, this interaction had been shown proteins into the ER in vivo: we observed no ERj3 and hardly any only in vitro. Here we confirmed with a cell-based demonstration AQ2 in Sec632/2 cells, whereas the amounts of other resident ER that the product of the SEC61A1 gene is involved in ribosome 1966 Journal of Cell Science 125 (8)

Fig. 5. The transport of prion protein is affected by the lack of Sec63. Murine kidney Sec63+/+ and Sec632/2 cells were treated with digitonin for preparation of semi- permeabilized cells. The indicated precursor polypeptides were imported into the indicated ER fractions under co- translational transport conditions. The membranes were subjected to sequestration analysis, followed by SDS-PAGE and phosphorimaging. Only the areas of interest for single gels are shown. Glycosylation efficiency was calculated for the shown gel and is given as mature protein as a percentage of precursor plus mature protein. g, glycosylated polypeptide; p, precursor; PK, proteinase K; RM, rough microsomes; TX100, Triton X-100; *PrP precursor was efficiently ubiquitylated; the characteristic ladder of mono-, di- or triple-ubiquitylated PrP was observed (Rane et al., 2008). RMs were less efficient at glycosylation than semi- permeabilized cells; therefore, processing by signal peptidase can be seen in addition to glycosylation.

binding and in co-translational transport of soluble and two model TA proteins that can be delivered to the ER membrane membrane proteins into the ER. Our findings must be by the SRP and SR (Abell et al., 2004) or TRC and the TRC considered in the context of the existence of the SEC61A2 gene receptor (WRB) (Stefanovic and Hegde, 2007; Rabu et al., 2008; in human cells. The product of this gene shows 95% sequence Vilardi et al., 2011). Therefore, we propose that the previously identity to SEC61A1 and should not be depleted under our observed cross-links between TA proteins and subunits of the conditions of SEC61A1-siRNA-mediated gene silencing. Thus, Sec61 complex must have resulted from the close association of the SEC61A2 gene could not complement the loss of function of SR and the Sec61 complex rather than from a role of the Sec61 the SEC61A1 gene, either because the SEC61A2 gene is not complex in TA membrane insertion (Abell et al., 2003). We note

Journal of Cell Science expressed in HeLa cells to any extent or its product has a that our findings on TA biogenesis in mammalian cells are different function to the product of the SEC61A1 gene. We favor entirely consistent with the corresponding observations in yeast the first explanation because expressed sequence tagging and cells (Steel et al., 2002; Yabal et al., 2003). array databases, as well as our preliminary RT-PCR experiments, indeed suggest that the SEC61A2 gene is expressed at a very low The Sec62 protein plays a role in post-translational level, even after silencing of the SEC61A1 gene (data not shown). transport of presecretory proteins into the mammalian ER Our experimental approach also allowed us to directly address Here, we provide the first demonstration that the product of the the question of SEC61A1 gene involvement in post-translational human SEC62 gene is involved in protein transport into the protein transport into the mammalian ER. This kind of protein mammalian ER. We note that ppcecA can partially use the SRP– transport has gained interest recently in the context of the SR pathway when ER membranes are present during its synthesis biogenesis of TA membrane proteins and associated cytosolic and that its post-translational transport is SRP and SR factors. Here, we provide the first demonstration that the product independent (Schlenstedt et al., 1990). Therefore, the partial of the SEC61A1 gene also is involved in post-translational inhibition of ppcecA transport under co-translational conditions transport of signal peptide-containing precursor proteins into the and the substantial inhibition of its post-translational transport human ER. This transport mechanism was previously shown to suggest that only the SRP- and SR-independent transport of be ribonucleoparticle independent and to involve cytosolic ppcecA involves Sec62. These observations are consistent with molecular chaperones, such as Hsc70 plus Hsp40 (Schlenstedt corresponding observations in yeast (Lyman and Schekman, and Zimmermann, 1987; Mu¨ller and Zimmermann, 1987 Mu¨ller 1997; Plath et al., 1998), but also raise the question of how they and Zimmermann, 1988; Schlenstedt et al., 1990; Klappa et al., can be reconciled with the presence of a ribosome-binding site in 1994). Furthermore, our experiments represent the first cell-based mammalian Sec62 (Mu¨ller et al., 2010). We propose that the demonstration that the product of the SEC61A1 gene is not ribosome-binding site in Sec62 is involved in coordinating co- involved in post-translational integration of TA proteins into the and post-translational protein transport in the mammalian ER, mammalian ER membrane. We note that the latter was observed e.g. by preventing synthesis of precursor proteins near the Sec61 not only for cytb5, which can be delivered to the ER membrane complex, whereas the Sec61 complex is engaged in post- by Hsc70 plus Hsp40 (Abell et al., 2007; Rabu et al., 2008) and translational transport. However, many more precursor spontaneously inserts into the membrane (Brambillasca et al., polypeptides will have to be analyzed under these conditions to 2005; Brambillasca et al., 2006), but also for Sec61b and Syb2, deduce any general rules. Protein transport into the mammalian ER 1967

The Sec63 protein plays precursor-specific roles in gating that Sec63 can facilitate integration of the model plasma of the mammalian Sec61 complex membrane protein AQP2 into the ER membrane of semi- Our findings are also the first demonstration of a role for Sec63 in permeabilized cells and that AQP2 is present at reduced levels co-translational protein translocation into the mammalian ER, in Sec632/2 cells. We note that AQP2 levels are also reduced in and add to the observed defect in the biogenesis of certain plasma Prkcsh2/2 cells, but that transport of Sec63-dependent precursor membrane proteins in a Sec632/2 model of human polycystic proteins such as AQP2 into the ER is not affected by the absence liver disease (Fedeles et al., 2011). Sec63 plays a substrate- of PRKCSH (not shown). Thus, these results confirmed the specific role in the initial insertion of certain precursors into the previous suggestion that PRKCSH and Sec63 act at different Sec61 complex, an early step of translocation. We suggest that in stages in the biogenesis of proteins such as AQP2 and – by the transport of certain precursors, such as ppl, the signal peptides extrapolation – polycystin-1. are strong enough to trigger opening of the Sec61 channel, i.e. after priming by the ribosome (Kim et al., 2002), whereas other Materials and Methods precursors require additional action of Sec63 (or even Sec63 plus Mouse genetics BiP). At least in the first case, the cytosolic Brl domain of Sec63 Complete SEC63 deletion results in pre-implantation defects because we never observed Sec632/2 embryos, even at E7.0. Therefore, we generated a conditional appears to be involved. We note that an early role for Sec63 Sec63flox allele with loxP sites flanking exon 2 and a neo cassette flanked by FRT and BiP in co-translational translocation of the precursor sites inserted into IVS2 (Fedeles et al., 2011). Removal of exon 2 by Cre of dipeptidyl-aminopeptidase B into the ER, as well as a recombinase activity results in complete loss of function (as documented by flox/flox contribution of the cytosolic domain of Sec63, were previously northern and western blotting). Sec63 mice were crossed with the ImmortoMouse interferon-c inducible H-2Kb-tsA58 SV40 temperature-sensitive observed in yeast (Young et al., 2001). Of further note, an early transgene. Renal tubules (of TAL origin) from these mice were microdissected and and precursor-specific role has also been reported for the the cells cultured in DMEM/Ham’s F-12 medium (PAA, Pasching, Austria) with mammalian TRAM protein (Voigt et al., 1996) and TRAP 1% fetal bovine serum (FBS), 10 ml/ml insulin–transferrin–selenium-X, 3.4 mg/ml complex (Fons et al., 2003), which raises the question of whether 3,39,5-triiodo-L-thyronine, 50,000 Units penicillin and streptomycin, and 10–20 Units interferon-c under humidified conditions at 33˚Cin5%CO2. The resultant or not the same precursor polypeptides involve Sec63, TRAM ‘parental’ cells were converted to a null cell line ex vivo by transfection with a and TRAP. Cre-recombinase plasmid (where Cre was fused to GFP) followed by either FACS What could distinguish Sec63-dependent from Sec63- sorting or limiting dilution cloning to obtain several clones of null cells (Sec632/2). In order to confirm complete deletion of the protein, western independent precursor polypeptides with respect to the initial blotting with anti-Sec63 antiserum was performed. phase of translocation? One distinguishing factor could be the properties of the cleavable or non-cleavable signal peptides. Cell growth and analysis Compared with the cleavable signal peptides of the other tested HeLa cells (ATCC no. CCL-2) and NIH/3T3 (Irene Schulz, Homburg) cells were precursors (22–42 residues, four to eight charges), the cleavable cultured in DMEM (Gibco-Invitrogen, Karlsruhe, Germany) containing 10% FBS (Biochrom, Berlin, Germany) and 1% penicillin and streptomycin (PAA, Pasching, signal peptides of ppcecA (22 residues, one charge), pERj3 (22 Austria) in a humidified environment with 5% CO2 at 37˚C. Sec63 control cells residues, no charged side chain) and pPrP (22 residues, no (derived from the Sec63floxed mouse) and Sec63 null cells were cultured in charged side chain) are short and apolar. However, more DMEM/Ham’s F-12 medium (PAA) containing 1% FBS, 1% insulin–transferrin– precursor polypeptides and chimeric precursors will have to selenium-X, 10 IU/l interferon- c from mouse, 1 mg/l 3,39,5-triiodo-L-thyronine, and 1% penicillin and streptomycin under humidified conditions at 33˚Cin5% Journal of Cell Science be analyzed to deduce the underlying principles. Another CO2. Growth rates and viability were determined using a CountessH Automated distinguishing feature could be that some signal peptides insert Cell Counter (Invitrogen). Cell viability was also evaluated using Nuclear-IDTM into the Sec61 complex in loop conformation (Shaw et al., 1988) Blue/Green cell viability reagent (Enzo Life Sciences, Lausen, Switzerland) whereas others insert in a ‘head-on’ orientation that is later according to the manufacturer’s instructions. followed by a ‘flip-turn’ (Devaraneni et al., 2011). The latter Silencing of gene expression by siRNA might require a larger internal diameter of the Sec61 channel, i.e. For SEC61A1 silencing, 5.26105 HeLa cells were seeded per 6 cm culture dish wider opening of the lateral gate, and therefore, help from Sec63 in normal culture. The cells were transfected with SEC61A1 siRNA (or Sec63 plus BiP). We note that a role of Sec63 and BiP in (59-GGAAUUUGCCUGCUAAUCAtt-39; Applied Biosystems, Darmstadt, Sec61 channel opening was previously shown for the early phase Germany), SEC61A1-UTR siRNA (59-CACUGAAAUGUCUACGUUUtt-39; Applied Biosystems), or control siRNA (AllStars Negative Control siRNA; of post-translational protein transport into the yeast ER (Lyman Qiagen, Hilden, Germany) at a final concentration of 20 nM using HiPerFect and Schekman, 1997). However, this idea was subsequently Reagent (Qiagen) as described previously (Erdmann et al., 2011; Lang et al., disputed by a model translocation reaction that allowed precursor 2011). After 24 hours, the medium was changed and the cells were transfected a second time. SEC62 silencing was carried out according to the same protocol with movement through the yeast Sec61 complex in detergent solution the previously described siRNAs (Greiner et al., 2011). For SEC63 gene silencing (Matlack et al., 1997). We suggest that in this experimental in HeLa or NIH/3T3 cells, 66105 cells were seeded per 6 cm culture dish in system Sec61 channel gating might have been facilitated by the normal culture. The cells were transfected with human SEC63 siRNAs (SEC63:59- detergent. GGGAGGUGUAGUUUUUUUAtt-39, SEC63-UTR: 59-AGUCUAUGGUCCC- AGUAAAtt-39; Applied Biosystems), murine siRNA (Sec63:59-CCAGAA- Our observations are also relevant in the context of polycystic CGCGGAGCAAAUUtt-39; Applied Biosystems) or the same control siRNA as liver disease. Autosomal dominant polycystic liver disease above at a final concentration of 20 nM using HiPerFect. For both cell types, the (ADPLD) is a rare human disorder, characterized by the medium was changed after 24 hours and the cells were transfected a second time. progressive development of biliary epithelial liver cysts. On the Silencing was evaluated by RT-PCR and western blot analysis. genetic level, ADPLD is heterogeneous involving at least two Complementation analysis different genes: PRKCSH, encoding the b-subunit of glucosidase In order to rescue the phenotype of SEC61A1, SEC62 or SEC63 silencing in HeLa II, and SEC63 (Drenth et al., 2003; Li et al., 2003; Davila et al., cells, the SEC61A1, SEC62 and SEC63 cDNA was inserted into the multiple 2004). Using mutant mice, it was observed that loss-of-function cloning site (MCS) of a pCDNA3-internal ribosomal entry site (IRES)-GFP vector mutations in Prkcsh or Sec63 result in reduced levels of the that contained the cytomegalovirus (CMV) promoter, the MCS, the IRES, plus the green fluorescent protein (GFP) coding sequence. Cells were treated with the plasma membrane protein polycystin-1 and, therefore, cyst respective UTR siRNA as described above for 48 hours. Subsequently, the cells formation in the liver (Fedeles et al., 2011). Here we showed were transfected with either vector or SEC61A1, SEC62 or SEC63 expression 1968 Journal of Cell Science 125 (8)

plasmid using Fugene HD (Roche Diagnostics, Mannheim, Germany). code for glycosylatable cytb5–ops28 and MHC class II inavariant Transfection efficiency was ,80%, as determined by GFP fluorescence. chain, Sec61b–ops13, synaptobrevin2–ops13 and aquaporin 2, respectively. The antibodies against Sec61c were a kind gift from Quantitative RT-PCR analysis K. Kapp and B. Dobberstein (Heidelberg). We thank Monika Lerner Cells were harvested and mRNA was isolated using the NucleoSpin RNA II Kit and Anika Mu¨ller (Homburg) for excellent technical assistance and (Machery and Nagel, Du¨ren, Germany). Reverse transcription was performed with Superscript II (Invitrogen) and oligo(dT) primer (Eurofins MWG Operon, Detlef Hof for writing the software that was used for EM analysis. Ebersberg, Germany). TaqManH Gene Expression Assays (Applied Biosystems) were used for quantitative real-time PCR of SEC61A1 (Hs00273698_m1), SEC61B Funding (Hs00606455_m1), SEC63 (Hs00273093_m1), ERJ1 (Hs01550849_m1) and BIP This work was supported by grants from the Deutsche (Hs99999174_m1) in the HT7900 system (Applied Biosystems). Dct values were Forschungsgemeinschaft [grant numbers GRK 845 to R.Z. and calculated using SOX (Hs01053049_s1) and BMI-1 (Hs00180411_m1) as a standard and the values were then normalized to control-siRNA-treated cells. FOR 967 to J.D. and R.Z.].

Semi-quantitative western blot analysis Supplementary material available online at Rabbit antibodies were raised against the C-terminal peptides of human Sec61a http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.096727/-/DC1 (14-mer), Sec62 (11-mer) and SRb (12-mer) or the N-terminal peptides of human BiP (12-mer), ERj3 (15-mer) and Grp170 (11-mer) plus an N- or C-terminal References cysteine, and against purified PDI, calreticulin and ERj1C-DN21 (Dudek et al., Abell, B. M., Jung, M., Oliver, J. D., Knight, B. C., Tyedmers, J., Zimmermann, R. 2005). Antibodies directed against AQP2 (New England Biolabs, Schwalbach, and High, S. (2003). Tail-anchored and signal-anchored proteins utilize overlapping Germany), calnexin (Stressgene, Hamburg, Germany), b-actin (Santa Cruz pathways during membrane insertion. J. Biol. Chem. 278, 5669-5678. Biotechnology, Heidelberg, Germany; Sigma, Taufkirchen, Germany), presenilin Abell, B. M., Pool, M. R., Schlenker, O., Sinning, I. and High, S. (2004). Signal 1 (Millipore, Eschborn, Germany) and SERCA2 (Sigma) were obtained from TM recognition particle mediates post-translational targeting in eukaryotes. EMBO J. 23, commercial sources. The primary antibodies were visualized using ECL Plex 2755-2764. TM goat anti-rabbit IgG–Cy5 conjugate or ECL Plex goat anti-mouse IgG–Cy3 Abell, B. M., Rabu, C., Leznicki, P., Young, J. C. and High, S. (2007). Post- conjugate (GE Healthcare, Freiburg, Germany) and the Typhoon-Trio imaging translational integration of tail-anchored proteins is facilitated by defined molecular system (GE Healthcare) in combination with Image Quant TL software 7.0 (GE chaperones. J. Cell Sci. 120, 1743-1751. Healthcare). Becker, T., Bhushan, S., Jarasch, A., Armache, J. P., Funes, S., Jossinet, F., Gumbart, J., Mielke, T., Berninghausen, O., Schulten, K. et al. (2009). Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating Immunofluorescence microscopy ribosome. Science 326, 1369-1373. Cells were fixed with paraformaldehyde, washed and an indirect Borgese, N. and Fasana, E. (2011). Targeting pathways of C-tail-anchored proteins. immunofluorescence staining was performed with an affinity purified rabbit Biochim. Biophys. Acta 1808, 937-946. antipeptide antibody directed against the C-terminal undecapeptide of human Brambillasca, S., Yabal, M., Soffientini, P., Stefanovic, S., Makarow, M., Hegde, Sec62 protein (plus an N-terminal cysteine) and Alexa-Fluor-488- or Alexa-Fluor- R. S. and Borgese, N. (2005). Transmembrane topogenesis of a tail-anchored protein 594-coupled secondary antibody from goat (Invitrogen). We note that the anti- is modulated by membrane lipid composition. EMBO J. 24, 2533-2542. Sec62 antibody is specific for Sec62 under denaturing as well as native conditions Brambillasca, S., Yabal, M., Makarow, M. and Borgese, N. (2006). Unassisted (i.e. western blot and fluorescence microscopy signals were quenched after translocation of large polypeptide domains across phospholipid bilayers. J. Cell Biol. silencing of the SEC62 gene) (Greiner et al., 2011). Cells were analyzed by 175, 767-777. microscopy using an Elyra SIM (Carl Zeiss MicroImaging, Go¨ttingen, Germany). Brodsky, J. L., Goeckeler, J. and Schekman, R. (1995). BiP and Sec63p are required Synthesis of the Sec62 protein was not affected by SEC61A1 silencing for 96 hours for both co- and posttranslational protein translocation into the yeast endoplasmic (supplementary material Table S1). reticulum. Proc. Natl. Acad. Sci. USA 92, 9643-9646. Colombo, S. F., Longhi, R. and Borgese, N. (2009). The role of cytosolic proteins in the insertion of tail-anchored proteins into phospholipid bilayers. J. Cell Sci. 122, Electron microscopy 2383-2392. Journal of Cell Science Cells were plated on poly-lysine-coated sapphire discs in 6-cm dishes and treated Davila, S., Furu, L., Gharavi, A. G., Tian, X., Onoe, T., Qian, Q., Li, A., Cai, Y., for 96 hours as described above. Sapphire discs were frozen in a high-pressure Kamath, P. S., King, B. F. et al. (2004). Mutations in SEC63 cause autosomal freezer and subjected to freeze substitution and embedding as previously described dominant polycystic liver disease. Nat. Genet. 36, 575-577. (Liu et al., 2010). Ultrathin (70 nm) sections were cut parallel to the cell Devaraneni, P. K., Conti, B., Matsumura, Y., Yang, Z., Johnson, A. E. and Skach, monolayer, collected on copper grids, stained with uranyl acetate and lead citrate, W. R. (2011). Stepwise insertion and inversion of a type II signal anchor sequence in and analyzed on the Tecnai 12 Biotwin electron microscope (Philips, Eindhoven, the ribosome-Sec61 complex. Cell 146, 134-147. The Netherlands). 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H., te Morsche, R. H. M., Smink, R., Bonifacino, J. S. and Jansen, transport experiment). Alternatively, precursor polypeptides were synthesized in J. B. M. J. (2003). Germline mutations in PRKCSH are associated with autosomal reticulocyte lysate in the presence of [35S]methionine for 15 minutes at 30˚C. After dominant polycystic liver disease. Nat. Genet. 33, 345-347. 5 minutes of incubation with RNase A (final concentration: 80 mg/ml) and Dudek, J., Greiner, M., Mu¨ller, A., Hendershot, L. M., Kopsch, K., Nastainczyk, W. cycloheximide (final concentration: 100 mg/ml) at 30˚C, buffer or semi- and Zimmermann, R. (2005). ERj1p has a basic role in protein biogenesis at the permeabilized cells were added and the incubation was continued for 30 endoplasmic reticulum. Nat. Struct. Mol. Biol. 12, 1008-1014. minutes (post-translational transport experiment). 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Triton X-100 was present at a final concentration of 1%. Protease treatment was Fons, R. D., Bogert, B. A. and Hegde, R. S. (2003). Substrate-specific function of the terminated by the addition of phenylmethylsulfonyl fluoride (10 mM). All samples translocon-associated protein complex during translocation across the ER membrane. were analyzed by SDS-PAGE and phosphorimaging (Typhoon-Trio imaging J. Cell Biol. 160, 529-539. system). Image Quant TL software 7.0 was used for quantifications. Go¨rlich, D. and Rapoport, T. A. (1993). Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell Acknowledgements 75, 615-630. Go¨rlich, D., Prehn, S., Hartmann, E., Kalies, K. U. and Rapoport, T. A. (1992). A We are grateful to N. Borgese (Milan, Italy), S. High (Manchester, mammalian homolog of SEC61p and SECYp is associated with ribosomes and UK) and W. 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