Distinct Roles for the Yeast Phosphatidylinositol 4-Kinases, Stt4p and Pik1p, in Secretion, Cell Growth, and Organelle Membrane Dynamics

Abstract

The yeast Saccharomyces cerevisiae possesses two genes that encode phosphatidylinositol (PtdIns) 4-kinases, STT4 and PIK1. Both gene products phosphorylate PtdIns at the D-4 position of the inositol ring to generate PtdIns(4)P, which plays an essential role in yeast viability because deletion of either STT4 or PIK1 is lethal. Furthermore, although both enzymes have the same biochemical activity, increased expression of either kinase cannot compensate for the loss of the other, suggesting that these kinases regulate distinct intracellular functions, each of which is required for yeast cell growth. By the construction of temperature-conditional single and double mutants, we have found that Stt4p activity is required for the maintenance of vacuole morphology, cell wall integrity, and actin cytoskeleton organization. In contrast, Pik1p is essential for normal secretion, Golgi and vacuole membrane dynamics, and endocytosis. Strikingly, pik1(ts) cells exhibit a rapid defect in secretion of Golgi-modified secretory pathway cargos, Hsp150p and invertase, whereas stt4(ts) cells exhibit no detectable secretory defects. Both single mutants reduce PtdIns(4)P by approximately 50%; however, stt4(ts)/pik1(ts) double mutant cells produce more than 10-fold less PtdIns(4)P as well as PtdIns(4,5)P(2). The aberrant Golgi morphology found in pik1(ts) mutants is strikingly similar to that found in cells lacking the function of Arf1p, a small GTPase that is known to regulate multiple membrane trafficking events throughout the cell. Consistent with this observation, arf1 mutants exhibit reduced PtdIns(4)P levels. In contrast, diminished levels of PtdIns(4)P observed in stt4(ts) cells at restrictive temperature result in a dramatic change in vacuole size compared with pik1(ts) cells and persistent actin delocalization. Based on these results, we propose that Stt4p and Pik1p act as the major, if not the only, PtdIns 4-kinases in yeast and produce distinct pools of PtdIns(4)P and PtdIns(4,5)P(2) that act on different intracellular membranes to recruit or activate as yet uncharacterized effector proteins.

Phosphoinositides in Constitutive Membrane Traffic

“…The mammalian ortholog of STT4 is PtdIns4KIII␣ (251,391) and the ortholog of PIK1 is PtdIns4KIII␤ (12,233,250). In yeast, STT4 and PIK1 do not compensate for each other in deletion studies, with STT4 functioning in regulation of the actin cytoskeleton and PIK1 essential for membrane traffic (9,129,370). PtdIns4KIII␤ has been localized to the Golgi, a location consistent with the work on yeast PIK1 (120,392).…”

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

“…Similarly, we found that overexpression of PIK1 could restore the sporulation of sec14-1 diploids to near wild-type frequency, and suppression of the meiotic defect was dependent on Spo14p (Table 2, lines 19 -20). In contrast, overexpression of the other PtdIns 4-kinase isoform, Stt4p, which supplies PtdIns(4)P primarily at the plasma membrane (Audhya et al, 2000), was unable to suppress the sporulation defect of sec14-1 cells (Table 2, line 21). These results support the hypothesis that PtdIns delivery is an essential function of Sec14p for spore formation and suggest that it is specifically the pool of PtdIns(4)P available at the Golgi that is critical.…”

“…Exit of secretory proteins from the Golgi requires the function of Sec14p, a PtdIns/PtdCho transfer protein (Bankaitis et al, , 1990, and Pik1p, a PtdIns 4-kinase, which phosphorylates PtdIns to synthesize PtdIns(4)P (Hama et al, 1999;Walch-Solimena and Novick, 1999;Audhya et al, 2000). Consequently, SEC14 and PIK1 are essential genes in vegetatively growing cells.…”

“…Consequently, SEC14 and PIK1 are essential genes in vegetatively growing cells. A second PtdIns 4-kinase, Stt4p (Yoshida et al, 1994), generates a distinct and essential pool of PtdIns(4)P (Audhya et al, 2000), whereas a third PtdIns 4-kinase (Lsb6p) has no essential function and is not required for sporulation (Han et al, 2002). There is substantial evidence that Sec14p contributes significantly to the pool of PtdIns that is converted to PtdIns(4)P in the Golgi via the action of Pik1p (Hama et al, 1999;Rivas et al, 1999).…”

Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

“…Mutants with defects in PI(4)P production by these lipid kinases showed reduced secretion, delayed vacuolar transport, defective protein retrieval from endosomes to the Golgi, and aberrant Golgi morphology (Godi et al, 1999;Hama et al, 1999;Walch-Solimena and Novick, 1999;Audhya et al, 2000). Recent work, mainly in mammalian cells, has revealed effectors of phosphatidylinositol 4-phosphate [PI(4)P] such as the PI(4)P adaptor proteins FAPP1 and FAPP2, which are involved in the generation of transport carriers for exocytosis (Godi et al, 2004;Vieira et al, 2005) and the clathrin adaptor AP-1 (Wang et al, 2003;Heldwein et al, 2004) that participates in the formation of clathrin-coated vesicles.…”