Open AccessOverexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels
Author(s) -
Indrani Mukherjee,
Charles Barlowe
Publication year - 2016
Publication title -
molecular biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.463
H-Index - 225
eISSN - 1939-4586
pISSN - 1059-1524
DOI - 10.1091/mbc.e15-10-0704
Subject(s) - copii , microbiology and biotechnology , biology , golgi apparatus , vesicle , rab , vesicle fusion , endoplasmic reticulum , calcium , vesicular transport proteins , secretory pathway , exocytosis , lipid bilayer fusion , transport protein , cytosol , gtpase , intracellular , secretion , biochemistry , chemistry , synaptic vesicle , membrane , endosome , enzyme , vacuolar protein sorting , organic chemistry
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.