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Formation of AP-3 transport intermediates requires Vps41 function

Nature Cell Biology volume 1, pages 346–353 (1999)Cite this article

Abstract

Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50–130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 δ-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.

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Figure 1: Enrichment and co-localization of ALP pathway cargoes and AP-3.
Figure 2: Immunoisolation of an ALP pathway membrane intermediate from Accudenz gradient fractions.
Figure 3: Morphology and immunocytochemistry of immunoisolated vesicles from vam3 tsf cells.
Figure 4: Enrichment of AP-3-coated vesicles by sucrose-gradient fractionation.
Figure 5: Biochemical analysis of VPS41 and AP-3 mutant cells.
Figure 6: Formation of the vesicular fraction depends on AP-3 or Vps41 function.
Figure 7: Vps41 binds to Apl5 in vivo and in vitro .
Figure 8: Vps41 functions together with AP-3 in the ALP pathway.

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Acknowledgements

We thank T. McQuistan and J.M. McCaffery for expert assistance with electron microscopy analysis. Affinity-purified Kex2 antibodies were a gift from G. Payne. We thank members of the Emr laboratory for helpful discussions, particularly A. Wurmser, J. Gary and M. Babst for critical reading of the manuscript. P.R. is supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Re 1384/1-1) and D.J.K. is an Associate of the Howard Hughes Medical Institute. This work was supported by NIH grant CA58689 (to S.D.E.). S.D.E. is an Investigator with the Howard Hughes Medical Institute.

Correspondence and requests for materials should be addressed to S.D.E.

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  1. Peter Rehling and Tamara Darsow: These authors contributed equally to this work

Authors and Affiliations

  1. Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, 92093-0668, , California, USA

    Peter Rehling, Tamara Darsow, David J. Katzmann & Scott D. Emr

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Correspondence to Scott D. Emr.

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Rehling, P., Darsow, T., Katzmann, D. et al. Formation of AP-3 transport intermediates requires Vps41 function. Nat Cell Biol 1, 346–353 (1999). https://doi.org/10.1038/14037

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