Analysis of Sec61p and Ssh1p interactions in the ER membrane using the split-ubiquitin system

BMC Cell Biology

Karin Römisch²  Carol Harty¹ 

[1] Current address: Sauder School of Business, Henry Angus Building, 2053 Main Mall, University of British Columbia, Vancouver, BC V6T 1Z2, Canada;Department of Microbiology, Faculty of Biology, Saarland University, Campus A1.5, Saarbruecken, 66123, Germany

关键词: Split-ubiquitin system;    Sec61 channel;    Endoplasmic Reticulum;    Protein translocation;   

Others  :  855430
DOI  :  10.1186/1471-2121-14-14

 received in 2012-12-31, accepted in 2013-02-28,  发布年份 2013

【 摘 要 】

Background

The split-ubiquitin system monitors interactions of transmembrane proteins in yeast. It is based on the formation of a quasi-native ubiquitin structure upon interaction of two proteins to which the N- and C-terminal halves of ubiquitin have been fused. In the system we use here ubiquitin formation leads to proteolytic cleavage liberating a transcription factor (PLV) from the C-ubiquitin (C) fusion protein which can then activate reporter genes. Generation of fusion proteins is, however, rife with problems, and particularly in transmembrane proteins often disturbs topology, structure and function.

Results

We show that both the Sec61 protein which forms the principal protein translocation channel in the endoplasmic reticulum (ER) membrane, and its non-essential homologue, Ssh1p, when fused C-terminally to CPLV are inactive. In a heterozygous diploid Sec61-CPLV is present in protein translocation channels in the ER membrane without disturbing their function and displays a limited set of protein-protein interactions similar to those found for the wildtype protein using biochemical methods. Although its expression level is similar, Ssh1-CPLV interactions are less strong, and, in contrast to Sec61p, Ssh1p does not distinguish between Sbh1p and Sbh2p. We show that interactions can be monitored by reporter gene activity or directly by PLV cleavage, which is more sensitive, but leads to quantitatively different results.

Conclusions

We conclude that the split-ubiquitin system we used here has high fidelity, but low sensitivity and is of limited use for detection of new, transient interactions with protein translocation channels in the ER membrane.

【 授权许可】

   
2013 Harty and Römisch; licensee BioMed Central Ltd.

【 图 表 】

Figure 3.

【 参考文献 】

• [1]Johnson AE, van Waes MA: The translocon: a dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 1999, 15:799-842.
• [2]Kalies KU, Allan S, Sergeyenko T, Kröger H, Römisch K: The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane. EMBO J 2005, 24(13):2284-2293.
• [3]Römisch K: Endoplasmic reticulum-associated degradation. Annu Rev Cell Dev Biol 2005, 21:435-456.
• [4]Finke K, Plath K, Panzner S, Prehn S, Rapoport TA, Hartmann E, Sommer T: A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae. EMBO J 1996, 15(7):1482-1494.
• [5]Wilkinson BM, Tyson JR, Stirling CJ: Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum. Dev Cell 2001, 3:401-409.
• [6]Wittke S, Dünnwald M, Albertsen M, Johnsson N: Recognition of a subset of signal sequences by Ssh1p, a Sec61p-related protein in the membrane of endoplasmic reticulum of yeast Saccharomyces cerevisiae. Mol Biol Cell 2002, 13:2223-2232.
• [7]Ng W, Sergeyenko T, Zeng N, Brown JD, Römisch K: Characterization of the proteasome interaction with the Sec61 channel in the endoplasmic reticulum. J Cell Sci 2007, 120(PT 4):682-691.
• [8]Johnsson N, Varshavsky A: Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci 1994, 91:10340-10344.
• [9]Varshavsky A: Three decades of studies to understand the functions of the ubiquitin family. Methods Mol Biol 2012, 832:1-11.
• [10]Stagljar I, Korostensky C, Johnsson N, Te Heesen S: A generic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci 1998, 95:5187-5192.
• [11]Stagljar I, te Heesen S: Detecting interactions between membrane proteins in vivo using chimeras. Methods Enzymol 2000, 327:190-198.
• [12]Wittke S, Lewke N, Müller S, Johnsson N: Probing the molecular environment of membrane proteins in vivo. Mol Biol Cell 1999, 10:2519-2530.
• [13]Yan A, Wu E, Lennarz WJ: Studies of yeast oligosaccharyl trasnferase subunits using the split-ubiquitin system: topological features and in vivo interactions. Proc Natl Acad Sci 2005, 102(20):7121-7126.
• [14]Yan A, Lennarz WJ: Two oligosaccharyl transferase complexes exist in yeast and associate with two different translocons. Glycobiology 2005, 15:1407-1415.
• [15]Chavan M, Yan A, Lennarz WJ: Subunits of the translocon interact with components of the oligosaccharyl transferase complex. J Biol Chem 2005, 280(24):22917-22924.
• [16]Mohorko E, Glockshuber R, Aebi M: Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation. J Inherit Metab Dis 2011.
• [17]Wheeler MC, Gekakis N: Defective ER associated degradation of a model luminal substrate in yeast carrying a mutation in the 4th ER luminal loop of Sec61p. Biochem Biophys Res Comm 2012, 427:768-773.
• [18]Carvalho P, Stanley AM, Rapoport TA: Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 2010, 143(4):579-591.
• [19]Van den Berg B, Clemons WM Jr, Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA: X-ray structure of a protein-conducting channel. Nature 2004, 427:36-44.
• [20]Hanein D, Matlack KE, Jungnickel B, Plath K, Kalies KU, Miller KR, Rapoport TA, Akey CW: Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 1996, 87(4):721-732.
• [21]Scheper W, Thaminy S, Kais S, Stagljar I, Römisch K: Coordination of N-Glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein. J Biol Chem 2003, 278(39):37998-38003.
• [22]Ng DT, Brown JD, Walter P: Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 1996, 134(2):269-278.
• [23]Pilon M, Römisch K, Quach D, Schekman R: Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane. Mol Biol Cell 1998, 9:3455-3473.
• [24]Young CL, Raden DL, Caplan JL, Czymmek KJ, Robinson AS: Cassette series designed for live-cell imaging of proteins and high-resolution techniques in yeast. Yeast 2012, 29:119-136.
• [25]Frauenfeld J, Gumbart J, Sluis EO, Gartmann M, Beatrix B, Mielke T, Berninghausen O, Becker T, Schulten K, Beckmann R: Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Nat Struct Mol Biol 2011, 18(5):614-621.
• [26]Toikkanen J, Gatti E, Takei K, Saloheimo M, Olkkonen VM, Söderlund H, de Camilli P, Keränen S: Yeast protein translocation complex: Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61ß subunit. Yeast 1996, 12:425-438.
• [27]Soromani C, Zeng N, Hollemeyer K, Heinzle E, Klein MC, Tretter T, Seaman MNJS, Römisch K: N-acetylation and phosphorylation of Sec complex subunits in the ER membrane. BMC Cell Biol 2012. in press
• [28]Jiang Y, Cheng Z, Mandon EC, Gilmore R: An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation. J Cell Biol 2008, 180:1149-1161.
• [29]Misselwitz B, Staeck O, Matlack KE, Rapoport TA: Interaction of BIP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex. J Biol Chem 1999, 274(29):20110-20115.
• [30]Young BP, Craven RA, Reid PJ, Willer M, Stirling CJ: Sec63p and Kar2p are required for the translocation of SRP-dependent precursors into the yeast endoplasmic reticulum in vivo. EMBO J 2001, 20(1–2):262-271.
• [31]Willer M, Jermy AJ, Young BP, Stirling CJ: Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane. Yeast 2003, 20(2):133-148.
• [32]Lakkaraju AK, Thankappan R, Mary C, Garrison JL, Taunton J, Strub K: Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation. Mol Biol Cell 2012, 23(14):2712-2722.
• [33]Mades A, Gotthardt K, Awe K, Stieler J, Döring T, Füser S, Prange R: Role of human Sec63 in modulation the steady-state levels of multi-spanning membrane proteins. PLoS One 2012, 7:e49243.
• [34]Dünnwald M, Varshavsky A, Johnsson N: Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum. Mol Biol Cell 1999, 10:329-344.
• [35]Guthrie C, Fink GR (Eds): In Guide to yeast genetics and molecular biology. 1991, 194. [Methods Enzymol]
• [36]Toyn JH, Gunyuzlu PL, White WH, Thompson LA, Hollis GF: A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance. Yeast 2000, 16(6):553-560.
• [37]Verma R, Chen S, Feldman R, Schieltz D, Yates J, Dohmen J, Deshaies RJ: Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 2000, 10:3425-3439.