Different Effects of Sec61a, Sec62 and Sec63 Depletion on Transport of Polypeptides Into the Endoplasmic Reticulum of Mammalian Cells
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1958 Research Article Different effects of Sec61a, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum 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 gene in human cells affects co- and post-translational transport of presecretory proteins 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 protein. 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 signal peptide-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