Molecular Genetics of Intracellular Protein Sorting 
specific transport vesicles that ultimately must 
recognize and fuse with the appropriate target 
organelle, the vacuole. Therefore the list of po- 
tential activities and structures required for the 
sorting and transport of lysosomal/vacuolar pro- 
teins can easily accommodate the large number 
of gene products presently implicated by the ge- 
netic studies. 
Role for a Protein Kinase Complex 
An understanding of this protein sorting path- 
way and the individual activities of different VPS 
gene products is being facilitated by the molecu- 
lar isolation and characterization of these genes. 
Thus far, we have cloned and sequenced eight 
VPS genes. Comparison of the yeast VPS gene se- 
quences with other known mammalian genes has 
revealed several informative structural and func- 
tional similarities. Two of the genes, VPS\ 5 and 
VPS54, are of particular interest. The sequence of 
the VPSl 5 gene predicts a protein with the fol- 
lowing features: a consensus site for amino- 
terminal myristoylation, a region that shares sig- 
nificant sequence similarity with the family of 
Ser/Thr protein kinases, and a region of homol- 
ogy with the regulatory subunit of protein phos- 
phatase 2A (PP2A). The sequence similarity 
Vpsl 5 protein (Vpsl 5p) shares with Ser/Thr pro- 
tein kinases and the PP2A regulatory subunit 
raises the interesting possibility that protein 
phosphorylation/dephosphorylation may play an 
important role in the regulation of protein sort- 
ing events. 
To assess the functional significance of these 
sequence similarities, we used site-directed mu- 
tagenesis to change several highly conserved 
amino acid residues in the putative kinase do- 
main of the Vpsl 5p. Each of the mutations inacti- 
vates the complementing activity of the VPS15 
gene. The mutant cells exhibit extreme vacuolar 
protein sorting defects; amino acid changes in 
less highly conserved positions gave only weak 
protein sorting defects. 
These data suggest that the Vpsl 5p is an active 
kinase and that this kinase activity is required 
during some step in vacuolar protein sorting. 
Protein phosphorylation may act as a "molecular 
switch" in this protein sorting pathway by ac- 
tively diverting vacuolar hydrolases away from 
the default secretion path and toward the vacu- 
ole. One can imagine several points in the path- 
way that may need to be regulated precisely, such 
as the budding and transport of carrier vesicles or 
the recognition and fusion of these with the 
correct target organelle. 
Biochemical and genetic evidence indicate 
that the Vpsl 5 protein forms a complex (on the 
cytoplasmic face of the membrane) with another 
Vps protein, Vps34p. Overexpression of Vps34p 
suppresses the vacuolar protein sorting and 
growth defects caused by mutations within the 
kinase domain of Vpsl 5p, but will not suppress a 
null allele of VPSl 5. Therefore, Vps34p cannot 
bypass the cells' requirement for Vpsl5p. This 
genetic interaction between VPSl 5 and VPS54 is 
consistent with the observation that mutations in 
both the VPSl 5 and VT534 genes result in a com- 
mon set of phenotypes. 
The Vps34 protein shares sequence similarity 
with a mammalian gene recently identified in 
Mike Waterfield's laboratory that codes for the 
catalytic subunit of the phosphatidylinositol 
3-kinase (PI3-kinase). P13-kinase catalyzes the 
formation of PI3-phosphate, a rare membrane 
lipid that has been proposed to act as a second 
messenger in cell signaling. The enzyme appears 
to play an important role in cell proliferation and 
transformation. PI3-kinase activity has been 
shown to be associated with several cell surface 
protein-tyrosine kinase receptors (e.g., the PDGF 
[platelet-derived growth factor], insulin, and 
CSF-1 [colony-stimulating factor 1] receptors). 
The role of this lipid-modifying enzyme in pro- 
tein trafficking events is not yet clear. However, 
we are in the process of mutating several con- 
served sequence motifs to analyze the signifi- 
cance of this intriguing sequence similarity in 
Vps34 protein function. 
Among its functions, PI3-phosphate formation 
at localized sites in the membrane (adjacent to 
membrane receptor proteins) may facilitate vesi- 
cle formation/targeting required during protein 
transport to the lysosome from both the Golgi 
complex (delivery of newly synthesized lyso- 
somal hydrolases) and the cell surface (endocytic 
uptake and down-regulation of cell surface re- 
ceptors). The Vps 15 kinase may regulate the ac- 
tivity of Vps34p/PI3-kinase by phosphorylation 
of Vps34p. 
We recently developed an in vitro assay that 
reconstitutes intercompartmental protein trans- 
port to the yeast vacuole. We are now using this 
assay to assign the Vpsl 5 and Vps34 proteins to a 
specific step(s) in the reaction. 
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