title: Overexpression of Sly41 suppresses COPII vesicle-tethering deficiencies by elevating intracellular calcium levels.
abstract: SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca(2+)/Mn(2+) transporter and suggests that Sly41 influences cellular Ca(2+) and Mn(2+) homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca(2+) concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca(2+) on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER-Golgi transport.
authors.affiliation: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
authors.forename: Indrani
authors.initials: I
authors.lastname: Mukherjee
authors.affiliation: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 charles.barlowe@dartmouth.edu.
authors.forename: Charles
authors.initials: C
authors.lastname: Barlowe
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D022181:COP-Coated Vesicles / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D052139:Intracellular Calcium-Sensing Proteins / Q000378:metabolism*; D008561:Membrane Fusion; D011485:Protein Binding; D050600:SNARE Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D052139:Intracellular Calcium-Sensing Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; D002118:Calcium