Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking.
In a survey of yeast genomic sequences encoding calcium- and phospholipid-binding C2 domains, three homologous genes were identified that encode proteins that each have three C2 domains and an apparent transmembrane domain near the N terminus. The name tricalbins is suggested for these proteins, corresponding to the open reading frames YOR086c (TCB1), YNL087w (TCB2), and YML072c (TCB3). An antiserum was raised against the C-terminal portion of tricalbin 2 and used on Western blots to demonstrate that the corresponding protein is expressed in yeast and appears as a high-molecular-weight band at 130 kDa with smaller fragments at 39 kDa and 46 kDa. A fusion protein consisting of full length tricalbin 2 fused to the green fluorescent protein was expressed in cells and found to traffic from the cell surface to intracellular vesicles near the vacuole. A two-hybrid interaction screen with the C-terminal portion of tricalbin 2 indicated that tricalbin 2 binds the C-terminal portions of tricalbins 1 and 3 suggesting that the tricalbins may form heterodimers in vivo. Tricalbin 2 also interacted with the activation domain of the pleiotropic drug resistance transcription factor Pdr1p. Combinatorial disruptions of the tricalbin genes revealed that tcb2 single mutants or tcb1, tcb3 double mutants have an altered vacuole morphology and are hypersensitive to cycloheximide. A screen for single-copy suppressors of the cycloheximide sensitivity of tricalbin mutants yielded RSP5, which encodes a C2-domain-containing, ubiquitin-conjugating ligase essential for receptor-mediated and fluid phase endocytosis. The results suggest that the tricalbins function as multimers in membrane-trafficking events and may provide insights into the roles of multi-C2-domain proteins, such as the synaptotagmins, in other organisms.
Creutz
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Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA. creutz@virginia.edu
Snyder
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Schulz
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D020816:Amino Acid Motifs; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry; D017353:Gene Deletion; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D005810:Multigene Family; D009154:Mutation; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012440:Saccharomyces / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013045:Species Specificity; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C085480:PDR1 protein, S cerevisiae; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C487548:TCB1 protein, S cerevisiae; C487549:TCB2 protein, S cerevisiae; C487550:TCB3 protein, S cerevisiae; D015534:Trans-Activators; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D003513:Cycloheximide