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PROTEIN SORTING IN THE YEAST SECRETORY PATHWAY 
Scott D. Emr, Ph.D., Associate Investigator 
Dr. Emr's laboratory is using the yeast Saccharo- 
myces cerevisiae as a model genetic system to study 
protein sorting to the lysosome-like vacuole. The 
laboratory previously designed a gene fusion-based 
selection scheme that enabled them to isolate more 
than 600 yeast mutants that exhibit severe defects in 
vacuolar protein sorting. The recessive mutations in 
these mutants define more than 33 complementa- 
tion groups that exhibit defects in vacuolar protein 
sorting {vps mutants). Extensive genetic, biochemi- 
cal, and morphological characterization of the vps 
mutants indicates that many of the VPS genes en- 
code components of the protein-sorting machinery 
responsible for either the selective recognition and 
packaging of vacuolar hydrolases into carrier vesi- 
cles or the transport and fusion of such vesicles with 
the correct target membrane. The isolation and char- 
acterization of the genes affected in these mutants is 
providing new insights into the molecular mecha- 
nisms that control protein traffic in the eukaryotic 
secretory pathway. 
Characterization of VPS Genes Required 
for Vacuolar Protein Sorting 
Dr. Emr's laboratory has cloned and sequenced 
eight VPS genes. Comparison of the VPS gene se- 
quences with other known genes has revealed sev- 
eral informative structural and functional similari- 
ties. Much of the laboratory's recent efforts have 
been directed toward the characterization of VPSl 5 
and VPS34, two genes of particular interest. The se- 
quence of the VPS15 gene predicts a protein with 
the following features: a consensus site for amino- 
terminal myristoylation, a region that shares signifi- 
cant sequence similarity with the family of Ser/Thr 
protein kinases, and a region of homology with the 
regulatory subunit of protein phosphatase 2A 
(PP2A). The sequence similarity the Vps 15 protein 
(Vpsl5p) shares with Ser/Thr protein kinases and 
the PP2A regulatory subunit raises the possibility 
that protein phosphorylation/dephosphorylation re- 
actions may play an important role in the regulation 
of protein-sorting events. Mutagenesis of several 
highly conserved amino acid residues in the puta- 
tive kinase domain of the Vpsl5p inactivated the 
complementing activity of the VPS 15 gene. The mu- 
tant cells exhibit extreme vacuolar protein-sorting 
defects. Vacuolar hydrolases like carboxypeptid- 
ase Y (CPY) are missorted and secreted by these 
mutants. 
Vpsl5p is a phosphoprotein, and mutations in its 
kinase domain eliminate its phosphorylation. 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 phosphoryla- 
tion may act as a "molecular switch" in this protein- 
sorting pathway by actively diverting vacuolar hy- 
drolases away from the default secretion path and 
toward the vacuole. Several points in the pathway, 
such as the budding and transport of carrier vesicles 
or the recognition and fusion of these vesicles with 
the vacuole, may need to be regulated precisely. A 
block in any one step could lead to missorting and 
secretion of vacuolar hydrolases. 
Short carboxyl-terminal truncations of Vpsl5p 
(removing as little as 30 amino acids) result in a 
temperature-conditional defect in the delivery of 
CPY to the vacuole. CPY delivery is essentially wild- 
type at the permissive temperature but almost com- 
pletely blocked immediately after shifting cells to 
the restrictive temperature. Upon imposition of the 
temperature block, the mutant cells accumulate 
48 
