newly synthesized p2 CPY within a saturable intra- 
cellular compartment that is distinct from the vacu- 
ole. This compartment does not appear to represent 
an aberrant, dead-end structure, because the accu- 
mulated p2 CPY can be efficiently processed to its 
mature form following a shift back to the permissive 
temperature. The rapid and efficient reversal of the 
block indicates that CPY is present within a normal 
intermediate of the vacuolar protein transport path- 
way. Dr. Emr's laboratory is presently attempting to 
purify this compartment. 
The VPS15 und VPS34 Gene Products 
Form a Protein Complex 
The substrates and activators of the novel protein 
kinase Vpsl5p must be identified to determine its 
precise role in vacuolar protein sorting. A search for 
multicopy plasmid suppressors of specific vpsl5 
mutations revealed that vpsl5 kinase domain muta- 
tions are suppressed by the overproduction of 
Vps34p but not by the overproduction of other VPS 
gene products. In contrast, the vacuolar protein- 
sorting defects of strains in which the VPS 15 gene 
has been deleted are not suppressed by the over- 
production of Vps34p, demonstrating that the 
overproduction of Vps34p cannot bypass the re- 
quirement for Vpsl5p in vacuolar protein sorting. 
These genetic data indicate that Vpsl5p and 
Vps34p functionally interact in yeast cells, probably 
at a common step in the vacuolar protein-sorting 
process. Native immunoprecipitation and chemical 
crosslinking experiments have demonstrated that 
the proteins also physically interact and are copreci- 
pitated by antisera specific for either of the individ- 
ual proteins. The combined biochemical and ge- 
netic data therefore demonstrate that Vpsl5p and 
Vps34p act together within a hetero-oligomeric 
protein complex to facilitate yeast vacuolar protein 
delivery. 
VPS34 Encodes a Lipid Kinase 
Recent data have provided some exciting new in- 
sights into the role of the Vpsl5/Vps34 protein 
complex in the protein-sorting reaction. Vps34p 
shares significant sequence similarity with the cata- 
lytic subunit of mammalian phosphatidylinositol 
3-kinase (PI3-kinase). PI3-kinase phosphorylates 
the membrane lipid phosphatidylinositol (PI) and 
other more highly phosphorylated PI derivatives 
(PI P and PI-P2) at the D-3 position of the inositol 
ring. In mammalian cells, PI3-kinase associates with 
many signal-transducing receptor tyrosine kinases 
(e.g., the platelet-derived growth factor [PDGF], in- 
sulin, and colony-stimulating factor- 1 [CSF- 1 ] recep- 
tors) and is postulated to be involved in the genera- 
tion of key second messenger molecules important 
for regulating cell growth and proliferation. 
PI3-kinase activity is readily detected in wild-type 
yeast cell extracts, but extracts of yeast strains de- 
leted for the VPS34 gene exhibit undetectable 
levels of PI3-kinase activity. In addition, strains 
overproducing Vps34p show elevated levels of 
PI 3 -kinase activity. These data indicate that VPS34 
encodes a PI 3 -kinase activity in yeast and suggest 
that PI3-kinase activity is involved in regulating in- 
tracellular protein sorting. Vps34p-mediated phos- 
phorylation of membrane PI could serve as a signal 
that triggers the specific interaction, or stabiliza- 
tion, of other proteins required for transport to the 
vacuole, such as vesicle coat proteins. 
One possibility presently being tested is that the 
Vpsl 5p kinase directly regulates the lipid kinase ac- 
tivity of Vps34p through a specific protein phos- 
phorylation reaction. Vpsl5p kinase activity may 
itself be regulated by direct interaction with particu- 
lar transmembrane receptors, such as a CPY-specific 
receptor. In such a model, Vpsl5p and Vps34p ef- 
fectively act as components of a signal transduction 
complex that transduces the signal received by the 
membrane receptors into a second messenger 
(PI3-phosphate) that could trigger the action of as 
yet unknown effector proteins that direct protein 
sorting to the vacuole. 
Dr. Emr is also Professor of Cellular and Molec- 
ular Medicine at the University of California, San 
Diego, School of Medicine. 
Articles 
Herman, P.K., Stack, J.H., and Emr, S.D. 1991. A 
genetic and structural analysis of the yeast Vpsl 5 
protein kinase: evidence for a direct role of 
Vpsl5p in vacuolar protein delivery. EMBO f 
10:4049-4060. 
Paravicini, G., Horazdovsky, B.F., and Emr, S.D. 
1992. Alternative pathways for the sorting of solu- 
ble vacuolar proteins in yeast: a vps35 null mu- 
tant missorts and secretes only a subset of vacuo- 
lar hydrolases. Mol Biol Cell 3:415-427. 
Robinson, J. S., Graham, T.R., and Emr, S.D. 1991. A 
putative zinc finger protein, Saccharomyces cer- 
evisiae VpslSp, affects late Golgi functions re- 
quired for vacuolar protein sorting and efficient 
a-factor prohormone maturation. Mol Cell Biol 
11:5813-5824. 
CELL BIOLOGY AND REGULATION 49 
