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First published online 26 September 2006
doi: 10.1242/jcs.03188

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Fab1p and AP-1 are required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S. cerevisiae

¹ Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London, WC1E 6BT, UK
² Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK
³ Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (111K): [in a new window]   Fig. 1. AP-1 complex deletion mutants show fab1-like sorting defects. (A) Aberrant sorting of GFP-CPS in mutants lacking AP-1 complex components. Yeast strains expressing GFP-CPS were inspected by fluorescence and light microscopy as indicated. Wild-type and fab1 strain are included for comparison. Like fab1 cells, apl2, apl4 and aps1 cells clearly fail to sort GFP-CPS to the vacuole lumen. The sorting defect of apm1 and apm2 cells is less transparent than in the other AP-1 component deletion mutants, probably because of the wild-type vacuole morphology of these cells. Similar results were seen with GFP-Phm5p (data not shown). (B) Vacuole morphology of AP-1 complex component deletion mutants. Vacuole morphology of the AP-1 component deletion mutants was visualized by staining with FM4-64 as described in the Materials and Methods, and inspected by fluorescence and light microscopy as indicated. Wild-type and fab1 strains are included for comparison. Both apl2 and apl4 cells have an un-lobed vacuole. The other AP-1 complex component deletion mutants have wild-type vacuole morphology. (C) AP-1 complex component deletion mutants have acidified vacuoles. Yeast cells were stained with quinacrine as described in the Materials and Methods, and inspected by fluorescence and light microscopy as indicated. Wild-type and fab1 strains are included for comparison. Unlike fab1 cells, all AP-1 component mutants retain quinacrine, demonstrating that their vacuoles are acidified correctly.

View larger version (87K): [in a new window]   Fig. 2. Trafficking of GFP-CPS and GFP-Sna3p in wild-type fab1, apl2 and apl4 cells. (A) Yeast cells expressing UbGFP-CPS were inspected by fluorescence and light microscopy as indicated. In all cells UbGFP-CPS was trafficked to the vacuole lumen, confirming that, like fab1 cells, apl2 and apl4 cells have lost specifically the ability to traffic ubiquitylated cargoes. (B) Yeast cells expressing GFP-Sna3p were inspected by fluorescence and light microscopy as indicated. In all cells GFP-Sna3p was trafficked to the vacuole lumen, demonstrating that trafficking from the TGN to the vacuole by the MVB is intact in apl2 and apl4 cells.

View larger version (44K): [in a new window]   Fig. 3. Trafficking of GFP-CPS, UbGFP-CPS and GFP-Sna3p in vps27, vps4 and fab1/vps4 cells. Cells were transformed with plasmids for expressing GFP-CPS, UbGFP-CPS or GFP-Sna3p, grown to mid-exponential phase and inspected by light and fluorescence microscopy. In all three mutants cargo proteins predominantly localize to the swollen MVB characteristic of class-E mutants. Thus, unlike in AP-1 complex component and fab1 mutants, UbGFP-CPS and GFP-Sna3p do not traffic to the vacuole lumen in vps27, vps4 and fab1/vps4 cells.

View larger version (51K): [in a new window]   Fig. 4. Overexpression of Fab1p reverts the trafficking defects of apl2 and apl4 cells. (A) Overexpression of Fab1p in vac14 cells restores wild-type trafficking of GFP-CPS. vac14 cells were transformed with either empty plasmid, (YEplac181), or plasmid containing FAB1 (YEplac181-FAB1) as indicated, and plasmid expressing GFP-CPS (pUG34-CPS), and inspected by fluorescence and light microscopy. (B) GFP-CPS trafficking to the vacuole lumen is restored in apl2 and apl4 cells by overexpression of Fab1p. apl2 and apl4 cells were transformed with pUG34-CPS and empty plasmid (YCplac33) or a 2 µ plasmid containing FAB1 under its own promoter (pEMY105) (Cooke et al., 1998; Yamamoto et al., 1995) and inspected by fluorescence and light microscopy as indicated. Overexpression of Fab1p restored wild-type trafficking to approximately 50% of apl2 cells and to more than 90% of apl4 cells. (C) Overexpression of Fab1p had no effects in the vacuole morphology of apl2 and apl4 cells.

View larger version (55K): [in a new window]   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.

View larger version (72K): [in a new window]   Fig. 6. Chitin-ring staining is restored to chs6 cells by deletion of either AP-1 complex genes, FAB1 or VAC14. Yeast cells were fixed, stained with Calcofluor White and inspected by fluorescence microscopy as described in Materials and Methods. (A) Chitin rings are visible in wild-type, apl2, apl4, chs6, fab1 and vac14 cells, but not in chs6 cells as indicated. (B) Deletion of APL2, APL4, FAB1 or VAC14 in chs6 cells restores chitin-ring staining, implying that Fab1p and Vac14p are required for AP-1 function. (C) Re-introduction of wild-type genes into chs6 double mutants restores the chs6 phenotype; no chitin rings are seen in these strains.

View larger version (5K): [in a new window]   Fig. 7. Role of AP-1 and Fab1p in GFP-CPS trafficking. Potential AP-1-dependent trafficking steps are shown as bold arrows; adapted from Black and Pelham, and Hinners and Tooze (Black and Pelham, 2000; Hinners and Tooze, 2003). In wild type-cells cargo traffic to the EE, are ubiquitylated by Rsp5p and, on reaching the MVB, are sorted into ILVs. The MVB fuses with the vacuole, releasing vesicles and cargo into the vacuole lumen. In cells where AP-1 and Fab1p function is compromised, cargo spill over into the GGA pathway, bypassing the EE, and arrive at the MVB un-ubiquitylated. As a result, cargoes fail to be recognised by the MVB sorting machinery and are delivered to the limiting membrane of the vacuole. In the absence of AP-1 and Fab1p function some cargo could also be trafficked to the plasma membrane and be ubiquitylated by Rsp5p; this could explain why there is ubiquitylation of cargo proteins in fab1 cells (Katzmann et al., 2004; Reggiori and Pelham, 2002).