ADP-Ribosylation Factors Do Not Activate Yeast Phospholipase Ds but Are Required for Sporulation

Abstract

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

“…These Arf-GAPs, and perhaps other proteins, may act as integrators that decipher the relative levels of PtdCho, PtdOH, and phosphoinositides and thereby adjust the rate of vesiclemediated membrane trafficking accordingly. In support of these ideas is the requirement for Arf1p in sporulation, which occurs downstream of Spo14p-mediated PtdOH synthesis (Rudge et al, 1998a) and perhaps explains why yeast do not contain an Arf-activated PLD (Rudge et al, 1998a;Rudge and Engebrecht, 1999). Once vesicles are generated, there may be a specific requirement for Spo14p-generated PtdOH in vesicle fusion at the SPBs (our unpublished results).…”

“…SPO14 tagged with the influenza virus hemagglutinin (HA) epitope and expressed from its own promoter (pME940) is fully functional and was characterized previously (Rudge et al, 1998b). pKR466 (spo14::URA3, which removes 4014 base pairs of the 5052-base pair ORF) and pME913 (spo14::LEU2, which removes 4187 base pairs) were used to generate chromosomal spo14 disruption/deletions and have been described (Rose et al, 1995;Rudge et al, 1998a).…”

Erv14 family cargo receptors are necessary for ER exit during sporulation inSaccharomyces cerevisiae

“…It may be that Vps13p and Rcy1p are similarly involved in protein recycling during prospore membrane extension. The shape and size of prospore membranes in these mutants are reminiscent of those in arf1 cells (Rudge et al, 1998). Arf proteins are involved in several events in intracellular vesicle trafficking and a specific function of Arf1p in prospore membrane formation has been suggested by the finding that an arf1⌬ arf2⌬ double mutant harboring an arf1-myc allele arrests sporulation with small prospore membranes (Rudge et al, 1998).…”

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects

“…Indeed, PtdOH contributes an important, but undefined function for efficient protein transport from the endoplasmic reticulum to the mammalian Golgi complex (30)(31)(32). Yet, yeast PLD is insensitive to activation by ADP-ribosylation factor (33) and is nonessential for secretory pathway function. Thus, yeast and mammalian PLDs might execute distinct physiological functions.…”