New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast.
The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca(2+) influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca(2+) after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
D Christian
DC
Martin
Hyemin
H
Kim
Nancy A
NA
Mackin
Lymarie
L
Maldonado-Báez
Carlos C
CC
Evangelista
Veronica G
VG
Beaudry
Drew D
DD
Dudgeon
Daniel Q
DQ
Naiman
Scott E
SE
Erdman
Kyle W
KW
Cunningham
D001393:Azoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002175:Candida / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000187:drug effects / Q000502:physiology; D055106:Genome-Wide Association Study / Q000379:methods; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D001393:Azoles; C108765:CCH1 protein, S cerevisiae; D015220:Calcium Channels; C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium
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