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Fig. 5. In apl2-1 and apl4-1 mutants GFP-CPS is incorrectly directed. (A) Point mutations used to generate the apl2-1 and apl4-1 mutants. We used site-directed mutagenesis to mutate the clathrin-binding sites in APL2 and the putative clathrin-binding sites of APL4 to produce the mutants apl2-1 and apl4-1. The non-clathrin-binding apl2-1 mutant has been described previously (Yeung and Payne, 2001). We substituted alanines for the two DLL motifs at residues 657-659 and 661-663 in APL4 to generate the apl4-1 mutant. Both apl2-1 and apl4-1 mutants expressed as full-length proteins when assayed by western blotting (data not shown). (B,C) Aberrant vacuole morphology and CPS-trafficking cells containing the apl2-1 and apl4-1 mutants. apl2D cells were transformed with empty plasmid (YCplac111), or plasmids for APL2, or apl2-1 expression. The vacuole morphology of these cells was inspected by FM4-64 staining as indicated, and GFP-CPS trafficking was assayed in these strains as described previously. The apl2-1 mutant is unable to support either wild-type vacuole morphology or wild-type GFP-CPS trafficking. Similar results were seen for the apl4-1 mutant in apl4D cells.
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