The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways

doi:10.1074/jbc.M208191200

The Ccz1-Mon1 Protein Complex Is Required for the Late Step of Multiple Vacuole Delivery Pathways*

From the ^‡Department of Molecular, Cellular, and Developmental Biology and the Department of Biological Chemistry and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109

Abstract

Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Δ and ccz1Δ strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Δ andmon1Δ are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.

Footnotes

↵* This work was supported by National Institutes of Health Public Health Service Grant GM53396 (to D. J. K.), the Lewis E. and Elaine Prince Wehmeyer Trust (to C.-W. W.), and a research fellowship from the Science and Technology Agency of Japan (to J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Yeast Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
↵¶ To whom correspondence should be addressed: University of Michigan, Dept. of Molecular, Cellular and Developmental Biology, Ann Arbor, MI 48109-1048. Tel.: 734-615-6556; Fax: 734-647-0884; E-mail: klionsky@umich.edu.
Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M208191200
↵2 P. E. Stromhaug and D. J. Klionsky, unpublished data.
Abbreviations:

CPY

carboxypeptidase Y

ALP

alkaline phosphatase

Ape1

aminopeptidase I

CFP

cyan fluorescent protein

Cvt

cytoplasm to vacuole targeting

GFP

green fluorescent protein

prApe1

precursor aminopeptidase I

PVC

pre-vacuolar compartment

SMD

synthetic minimal medium with dextrose

SD/-N

synthetic minimal medium with dextrose but lacking nitrogen

YFP

yellow fluorescent protein

ORF

open reading frame

MES

4-morpholineethanesulfonic acid

PIPES

1,4-piperazinediethanesulfonic acid
- Received August 9, 2002.
- Revision received September 30, 2002.
The American Society for Biochemistry and Molecular Biology, Inc.