title: Analysis of Sec61p and Ssh1p interactions in the ER membrane using the split-ubiquitin system.
abstract: 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.
authors.affiliation: Cambridge Institute for Medical Research, Hills Road, Cambridge CB2 2XY, UK.
authors.forename: Carol
authors.initials: C
authors.lastname: Harty
authors.forename: Karin
authors.initials: K
authors.lastname: Römisch
mesh_terms: D004171:Diploidy; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D018139:Simplexvirus / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
chemical_list: D017405:Herpes Simplex Virus Protein Vmw65; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; C499381:SSH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin