Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast.

Abstract

Abstract. A membrane-associated complex composed of the Vpsl5 protein kinase and the Vps34 phosphatidylinositol 3-kinase (Ptdlns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vpsl5p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p Ptdlns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vpsl5 proteins. In contrast, catalytically inactive Vpsl5p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vpsl5p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperatureconditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular Ptdlns(3)P levels, indicates that the loss of Vpsl5p function leads to a defect in activation of Vps34p. In addition, characterization of a temperaturesensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of Ptdlns 3-kinase activity since cellular Ptdlns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular Ptdlns(3)P levels in various vps15 and vps34 mutant strains has led to additional insights into the importance of PtdIns(3)P intraceUular localization, as well as the roles of Vpsl5p and Vps34p in vacuolar protein sorting.

Phosphatidylinositol 3-Phosphate, an Essential Lipid in Plasmodium, Localizes to the Food Vacuole Membrane and the Apicoplast

“…Wortmannin is known to be unstable in complex media and at a neutral pH (66), and accordingly, we could not detect any effect of a single dose as high as 25 M wortmannin in a standard 2-day P. falciparum growth assay (10). However, the alternative PI3-kinase inhibitor LY294002 (59), which is stable under cell culture conditions, interfered with P. falciparum growth, with a 50% inhibitory concentration (IC 50 ) of 26 Ϯ 6 M (mean Ϯ SEM; n ϭ 4), indicating that PI3-kinase might also be essential for P. falciparum development.…”

Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy

“…The PtdIns 3-kinase complex I, which consists of Vps34, Vps15, Vps30/Atg6, and Atg14, has been shown to be required for all types of autophagy (Kihara et al, 2001). Indeed, by using strains lacking either Vps15 or Vps34 or a temperature-sensitive allele of Vps34 ( Figure 3A) (Stack et al, 1995), we were able to verify that PtdIns(3)P is required for the association of Atg18, Atg21, and Ygr223c with the vacuole.…”

Autophagic Processes in Yeast: Mechanism, Machinery and Regulation

“…PtdIns 3-kinase complex: In yeast there are at least two protein complexes that direct the synthesis of PtdIns3P (Kihara et al 2001) (Herman et al 1991;Stack et al 1993;Stack et al 1995;Kametaka et al 1998;Kihara et al 2001). Complex I also includes Atg14 and functions in autophagy, whereas complex II contains Vps38 and is involved in endosomal trafficking, endocytosis, and the vacuolar protein sorting pathway (Kihara et al 2001).…”