Human ARF4 expression rescues sec7 mutant yeast cells.
Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.
Deitz
S B
SB
Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Wu
C
C
Silve
S
S
Howell
K E
KE
Melançon
P
P
Kahn
R A
RA
Franzusoff
A
A
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D002352:Carrier Proteins / Q000096:biosynthesis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004790:Enzyme Induction; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D015723:Gene Library; D005816:Genetic Complementation Test; D006026:Glycoside Hydrolases / Q000096:biosynthesis; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*; D013489:Suppression, Genetic; D014407:Tumor Cells, Cultured; D043324:beta-Fructofuranosidase
D002352:Carrier Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; C066755:Sec7 guanine nucleotide exchange factors; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors