The C Terminus of the Vps34p Phosphoinositide 3-Kinase Is Necessary and Sufficient for the Interaction with the Vps15p Protein Kinase

Abstract

Vps34p is a phosphatidylinositol 3-kinase that is part of a membrane-associated complex with the Vps15p protein kinase. This kinase complex is required for the delivery of soluble proteins to the lysosomal/vacuolar compartment of eukaryotic cells. This study examined the Vps34p-Vps15p association and identified the domains within each protein that were important for this interaction. Using several different approaches, the interaction domain within Vps34p was mapped to a 28-amino acid element near its C terminus. This Vps34p motif was both necessary and sufficient for the interaction with Vps15p. Two-hybrid mapping experiments indicated that two separate regions of Vps15p were required for the association with Vps34p; they are the N-terminal protein kinase domain and a set of three tandem repeats of about 39 amino acids each. Neither domain alone was sufficient for the interaction. These Vps15p repeat elements are similar in sequence to the HEAT motifs that have been implicated in protein interactions in other proteins, including the Huntingtin protein. Finally, these studies identified a novel motif at the very C terminus of Vps34p that was required for phosphatidylinositol 3-kinase activity. This domain is highly conserved specifically in all Vps34p-like phosphatidylinositol 3-kinases but is not required for the interaction with Vps15p. This study thus represents a first step toward a better understanding of how this Vps15p⅐Vps34p kinase complex is assembled and regulated in vivo.

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

“…S1 in the supplemental material). Mutational analysis in S. cerevisiae revealed that the last amino acids of PI3-kinase are essential for kinase activity (6). On the other hand, Cterminal fusion proteins with GFP or the TAP tag have been described in the literature (24).…”

The Ras/cAMP-dependent Protein Kinase Signaling Pathway Regulates an Early Step of the Autophagy Process in Saccharomyces cerevisiae

“…For the rapamycin experiments, autophagy assays were performed on cells that had been treated with 0.2 g/ml rapamycin for 0, 1, or 3 h at 30°C. Western Immunoblotting and Immunoprecipitations-For the Western immunoblots, protein extracts were prepared by a glass-beading method described previously (56,57). The resulting protein extracts were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences) at 4°C.…”

“…The Supersignal chemiluminescent substrate (Pierce) was subsequently used to illuminate the reactive bands. The carboxypeptidase Y (CPY) immunoprecipitation experiments were performed as described (57,59).…”

Vps34p Is Required for the Decline of Extracellular Fructose-1,6-bisphosphatase in the Vacuole Import and Degradation Pathway

“…This function is conserved from yeast to human (44). For the Vps pathway, Vps34p is recruited from the cytosol to the Golgi/endosome via interaction with the membrane-associated protein kinase Vps15p, which also stimulates the phosphatidylinositol 3-kinase activity of Vps34p (48,49). The C-terminal 11 amino acids of Vps34p (amino acids 864 -875) are implicated in the protein association on the membrane (50).…”

“…The C-terminal 11 amino acids of Vps34p (amino acids 864 -875) are implicated in the protein association on the membrane (50). Furthermore, the deletion of the C-terminal 11 residues reduces PI3K activity (49,50).…”