title: The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B.
abstract: In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31. BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis. Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein. PTPLB turns over rapidly through degradation by the proteasome system. Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.
authors.affiliation: Department of Biochemistry. McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6.
authors.forename: Bing
authors.initials: B
authors.lastname: Wang
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.forename: Michel J
authors.initials: MJ
authors.lastname: Massaad
authors.forename: Annette
authors.initials: A
authors.lastname: Herscovics
authors.forename: Gordon C
authors.initials: GC
authors.lastname: Shore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D006836:Hydro-Lyases; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: C091247:BCAP31 protein, human; C489642:Bcap31 protein, mouse; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D017027:Protein Tyrosine Phosphatases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D006836:Hydro-Lyases; C484872:HACD2 protein, human