Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function.
A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.
Cheng
Hong
H
Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Japan.
Sugiura
Reiko
R
Wu
Wenlian
W
Fujita
Masaaki
M
Lu
Yabin
Y
Sio
Susie O
SO
Kawai
Rena
R
Takegawa
Kaoru
K
Shuntoh
Hisato
H
Kuno
Takayoshi
T
D000199:Actins / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002455:Cell Division / Q000502:physiology; D002473:Cell Wall / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016559:Tacrolimus / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D065095:Calcineurin Inhibitors; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C075976:ypt3 protein, S pombe; D019703:Calcineurin; D020691:rab GTP-Binding Proteins; D016559:Tacrolimus