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Published online 16 October 2000. doi:10.1083/jcb.151.2.297
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© The Rockefeller University Press, 0021-9525/2000//297 $5.00
The Journal of Cell Biology, Volume 151, Number 2, , 2000 297-310

Original Article

Sorting of Yeast Membrane Proteins into an Endosome-to-Golgi Pathway Involves Direct Interaction of Their Cytosolic Domains with Vps35p



Steven F. Nothwehra, Seon-Ah Haa, and Paul Bruinsmaa

a Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211
Division of Biological Sciences, 401 Tucker Hall, University of Missouri, Columbia, Missouri 65211.(573) 882-0123(573) 884-6461

Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar–endosomal compartment, a process dependent on aromatic amino acid–based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal–dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

Key Words: Saccharomyces cerevisiae • vps • protein sorting • endosome • Golgi

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AP, adaptor protein; CEN, yeast centromere; CPY, carboxypeptidase Y; DIC, differential interference contrast; DPAP, dipeptidyl aminopeptidase; DSP, dithiobis(succinimidylpropionate); M6P, mannose 6–phosphate; PVC, prevacuolar–endosomal compartment.


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