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© The Rockefeller University Press, 0021-9525/1997//1761 $5.00
The Journal of Cell Biology, Volume 139, Number 7, , 1997 1761-1774

Article

The Yeast Adaptor Protein Complex, AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole



J. David Stepp, Kristen Huang, and Sandra K. Lemmon

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-{Delta} 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-{Delta} 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.

Abbreviations used in this paper: *ALP, aberrantly processed ALP; ALP, alkaline phosphatase; AP, adaptor protein; CPY, carboxypeptidase Y; IP, immunoprecipitation; mALP, mature ALP; mts, apm three synthetic lethal mutant; pALP, precursor ALP; PIC, protease inhibitor cocktail; PVC, prevacuolar compartment; SNARE, soluble N-ethymaleimide-sensitive factor attachment protein receptor; ts, temperature sensitive; Vps, vacuolar protein sorting; wt, wild type.

Address all correspondence to Sandra K. Lemmon, Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4960. Tel.: (216) 368-6279. Fax: (216) 368-3055. E-mail: skl{at}po.cwru.edu

We are particularly indebted to S. Nothwehr (University of Missouri) for his generous supply of anti-alkaline phosphatase antibodies. M. Carlson (Columbia University), S. Emr (University of California, San Diego, CA), R. Fuller (University of Michigan), E. Jones (Carnegie Mellon University), P. Kane (SUNY Health Science Center at Syracuse, Syracuse, NY), D. Loyaza (Johns Hopkins University, Baltimore, MD), S. Michaelis (Johns Hopkins University), R. Piper (University of Iowa, Iowa City, IA), H. Riezman (University of Basel), L. Robinson (Louisiana State University, Shreveport, LA), T. Stevens (University of Oregon, Eugene, OR), and L. Weisman (University of Iowa) also generously provided strains, plasmids, and/or antibodies. S. Nothwehr, R. Piper, L. Conibear (University of Oregon), P. Kane, L. Robinson, and L. Weisman provided many helpful suggestions. We thank H. Riezman and G. Payne (University of California, Los Angeles, CA) for communicating results before publication. We thank G. Matera (all from case Western Reserve University) and members of his lab for providing frequent use of their Zeiss Axioplan, J. Polak and R.-Z. Wang for their assistance with EM and T. Nilsen for use of his phosphorimager. We also acknowledge E. Noss and M. Otsuka for their help in generation of some of the strains and plasmids used in these studies. Finally, we thank M. Snider, H. Riezman, and members of the Lemmon and Riezman labs for stimulating discussions and critical reading of this manuscript.


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