Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

Abstract

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of α-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and α-factor maturation and identify structural determinants that are critical for these functions.

ACKNOWLEDGMENTS

We thank Xiaolin Zhu for excellent technical assistance; Todd Graham, Sandra Lemmon, Tom Stevens, Carol Woolford, and Mark Rose for generous gifts of antibodies; and Tom Stevens and Neil Green for yeast strains. We thank Jose Martina for the gift of negative control GST fusions. We thank Cathy Jackson and Cecilia Bonangelino for critical reading of the manuscript.

C.M. is supported by a National Research Council Post-Doctoral Associateship.