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Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio
44106
A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast
and in animals. To gain insight into the role of yeast
AP-3, a genetic strategy was devised to isolate gene
products that are required in the absence of the AP-3 µ chain encoded by APM3. One gene identified by this
synthetic lethal screen was VPS45. The Vps pathway
defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is
transported via an alternate, intracellular route. This suggested that the apm3-
vps45 synthetic phenotype
could be caused by a block in both the alternate and the
Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing
and missorting of ALP. ALP is no longer localized to
the vacuole membrane by immunofluorescence, but is
found in small punctate structures throughout the cell.
This pattern is distinct from the Golgi marker Kex2p,
which is unaffected in AP-3 mutants. We also show that
in the apm3- mutant some ALP is delivered to the
vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way
to the vacuole or destined for recycling to the Golgi.
Surprisingly, in AP-3 class E vps double mutants these
proteins reappear on the vacuole. We suggest that some
AP-3-dependent cargo proteins that regulate late steps
in Golgi to vacuole transport are diverted into the Vps
pathway allowing completion of transfer to the vacuole
in the class E vps mutant.
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