Abstract
Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat-containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized γ-ear-binding sites found in other AP-1-binding proteins. This motif in Laa2 is essential for the Laa1-AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the γ-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.
Highlights
Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system
When intensity values of individual puncta were determined, those in laa2⌬ were on average half as intense as those in WT cells, and the maximum intensity of puncta in laa2⌬ cells was lower than the median intensity in WT cells. This defect was not due to a significant loss of Apl2 expression, as determined by Western blotting analysis, indicating a severe defect in AP-1 localization (Fig. 1C). To determine whether this effect was specific to AP-1, we examined the effect of laa2⌬ on the localization of Laa1 and on Gga2, a clathrin adaptor that localizes to the trans-Golgi network (TGN)
Proteins of the HEAT repeat– containing 5 (HEATR5) family are important for AP-1 function in diverse organisms (16, 19 –22, 36)
Summary
Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. HEATR5b binds two proteins, aftiphilin and ␥-synergin, that themselves interact directly with the ␥-ear [19, 23, 24]. This mutation in LAA2 did not alter the interaction of the ␥-ear with Ent3 or Ent5, clathrin adaptors that contain their own ␥-ear– binding motifs (Fig. 4, D and F) [34].
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