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Related Experiment Videos

Human ARF4 expression rescues sec7 mutant yeast cells

S B Deitz1, C Wu, S Silve

  • 1Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

Molecular and Cellular Biology
|July 1, 1996
PubMed
Summary

Human ADP ribosylation factor 4 (hARF4) rescues yeast sec7 mutants by restoring secretory pathway function. This suggests Sec7p requires cycling between cytosolic and membrane pools for vesicle traffic and cell growth.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Vesicle-mediated transport is crucial for the yeast secretory pathway, involving protein recruitment for budding, targeting, and fusion.
  • Sec7p, a protein in yeast transport vesicle coat structures, plays a role in Golgi-bound vesicle formation.
  • Understanding Sec7p function and interactions is key to deciphering mammalian secretory pathway regulation.

Purpose of the Study:

  • To identify mammalian homologs and interacting proteins of yeast Sec7p.
  • To investigate the role of human ADP ribosylation factor 4 (hARF4) in suppressing yeast sec7 mutations.
  • To elucidate the mechanism by which hARF4 influences Sec7p localization and secretory pathway function.

Main Methods:

  • Genetic selection using a human HepG2 cDNA library transformed into conditional-lethal yeast sec7 mutants.

Related Experiment Videos

  • Isolation and identification of cDNA clones that rescue sec7 mutant growth.
  • Analysis of Sec7p cytosolic and membrane-associated pools in the presence and absence of hARF4.
  • Investigating the effect of overexpressing yeast ARF1, ARF2, and YPT1 on sec7 mutant phenotype.

    • Main Results:

    • Human ADP ribosylation factor 4 (hARF4) was identified as a potent suppressor of the yeast sec7 mutation.
    • hARF4 restored secretory pathway function in sec7 mutants, indicating it interacts functionally with Sec7p.
    • hARF4 maintained the balance between cytosolic and membrane-associated Sec7p pools, which is essential for cell growth.
    • Overexpression of yeast ARF1 and ARF2, but not YPT1, also suppressed the sec7 mutation in an allele-specific manner.

    Conclusions:

    • hARF4 functions in the yeast secretory pathway by interacting with Sec7p, rather than being a direct homolog.
    • Sec7p cycling between cytosolic and membrane compartments is critical for its function in vesicular traffic and cell viability.
    • Specific yeast ARF proteins (ARF1, ARF2) are involved in distinct Sec7p-dependent functions within vesicle coat dynamics.