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doi: 10.1242/10.1242/jcs.00090

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Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase

Patricie Burda, Steven M. Padilla, Srimonti Sarkar and Scott D. Emr*

Department of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 92093-0668, USA



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  		Fig. 1. Vacuolar sorting of ALP and CPS is not affected in {Delta}vps30 and {Delta}vps38 mutant cells. (A) Wild-type (SEY6210), {Delta}vps30 (JCY300), {Delta}vps38 (PBY58) and {Delta}vps30{Delta}vps38 (PBY51) strains were grown in YNB medium to early log phase and metabolically labeled with [35S] methionine/cysteine at 26°C for 10 minutes and then chased in the presence of excess non-labeled amino acids for 30 minutes. The proteins were immunoprecipitated with antibodies specific to CPY, resolved on SDS-PAGE and visualized by autoradiography. The migration positions of Golgi-modified precursor (p2) and mature (m) CPY are shown. (B) Cells were grown in YPD medium to early log phase. Total cell lysates were subjected to SDS-PAGE, followed by immunoblotting with anti-CPS and anti-ALP antibodies, respectively. CPS samples were treated with endoglycosidase H prior to electrophoresis. Precursor forms of CPS and ALP are indicated (p; 73 kDa and 76 kDa, respectively) and mature forms (m; 69 kDa and 72 kDa, respectively) are shown. (C) Fluorescence and DIC microscopy of cells expressing GFP-CPS (pGO45).

 


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  		Fig. 2. The CPY sorting receptor Vps10p is mislocalized to the vacuolar membrane in {Delta}vps30 and {Delta}vps38 mutant strains. Wild-type (PBY34), {Delta}vps30 (PBY38) and {Delta}vps38 (PBY40) cells expressing Vps10-GFP were labeled in YPD with the fluorescent dye FM4-64 for 10 minutes at room temperature. The dye was washed away, the cells were chased for an additional 60 minutes in YPD and then put on ice. Localization of FM4-64 and Vps10-GFP were compared by fluorescence microscopy.

 


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  		Fig. 3. Vps30 and Vps38 protein functions are required for stability of the Golgi-localized Kex2 protease. Wild-type (SEY6210), {Delta}vps30 (JCY300), {Delta}vps38 (PBY58) and {Delta}vps35 (EMY18) cells carrying plasmid pPB1 (KEX2-HA) were grown to early log phase and labeled with [35S] methionine/cysteine at 26°C for 20 minutes followed by the addition of excess non-labeled amino acids (chase). Samples were removed at 0 and 90 minutes after the addition of chase. Kex2-HA (upper panel) was immunoprecipitated from cell lysates using HA-specific antibodies. The same cell lysates were then re-immunoprecipitated with G6PDH-specific antibodies (lower pannel). Kex2-HA and G6PDH were resolved by SDS-PAGE and visualized by autoradiography.

 


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  		Fig. 4. {Delta}vps30 and {Delta}vps38 mutant cells exhibit defects in PtdIns3P synthesis. Wild-type (SEY6210), {Delta}vps30 (JCY300), {Delta}vps38 (PBY58), and {Delta}vps30{Delta}vps38 (PBY51) cells were grown in YNB to early log phase and labeled with myo-[2-3H] inositol at 26°C for 45 minutes. Cellular lipids were recovered, deacylated and separated by HPLC. Levels of deacylated products corresponding to the indicated phosphoinositides are shown. These data represent the means±s.e.m. of at least three independent experiments. PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(3,5)P2, phosphatidylinositol (3,5)-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate.

 


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  		Fig. 5. Recruitment of Vps5p and Vps17p to the endosomal membrane depends on the synthesis of PtdIns3P by the Vps34p PtdIns 3-kinase complex II. (A) Localization of Vps5-GFP in wild-type (PBY86), {Delta}vps34 (PBY88) and {Delta}vps38 (PBY87) cells (left panel) and Vps17-GFP in wild-type (PBY70), {Delta}vps34 (PBY111) and {Delta}vps38 (PBY112) cells (right panel) by fluorescence microscopy. (B) Fluorescence and DIC microscopy of Vam7-GFP (pTKS35) expressed in wild-type (SEY6210) and {Delta}vps38 (PBY58) cells, respectively. (C) Fluorescence and DIC microscopy of vps34ts cells expressing Vps17-GFP (PBY123). The cells were incubated at non-permissive temperature (37°C) as indicated.

 


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  		Fig. 6. Proper localization of the CPY receptor Vps10p depends on Vps34p-mediated PtdIns3P synthesis. Fluorescence and DIC microscopy of vps34ts cells expressing Vps10-GFP (PBY124). The cells were incubated at non-permissive temperature (37°C) as indicated.

 


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  		Fig. 7. The PX domains of Vps5p and Vps17p, respectively, bind specifically to PtdIns3P. (A) The ability of the PX domains of Vps5p and Vps17p to bind lipids in vitro was tested by a protein-lipid overlay assay using GST-PX domain fusion proteins and nitrocellulose-immobilized phospholipid strips. PI, phosphatidylinositol; PC, phosphatidylcholine. (B) Serial dilutions of PtdIns3P (100 pmol, 50 pmol and 25 pmol) were spotted onto nitrocellulose membranes and incubated with 10 ng ml-1 of purified GST-Vps5 PX, GST-Vps17 PX or Vam7 PX domain fusion protein. (C) Localization of Vps17-GFP in PtdIns-kinase mutant strains defective for synthesis of PtdIns(3,5)P2 ({Delta}fab1, PBY116), PtdIns (4)P (pik1tsf, PBY114; stt4tsf, PBY115), and PtdIns (4,5)P2 (mss4tsf, PBY117). The strains were incubated at non-permissive temperature (37°C) as indicated.

 


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  		Fig. 8. The Vps5 PX domain is responsible for Vps5p targeting and functionality. Point mutations in conserved PX domain residues of Vps5p were introduced as described in Materials and Methods (Y322A, R360A). (A) Localization of wild-type Vps5-GFP (pPB2) or mutant vps5Y322A,R360A_ GFP fusion protein (pPB3) in {Delta}vps5 cells (BHY152). (B) Wild-type (SEY6210), {Delta}vps5 (BHY152) and {Delta}vps5 cells carrying plasmid pPB4 (vps5Y322A,R360A) were analyzed by pulse-chase labeling and immunoprecipitation with antibodies against CPY. The migration positions of ER-modified (p1), Golgi-modified precursors (p2) and mature (m) CPY are shown. (C) Protein-lipid overlay assay using nitrocellulose-immobilized phospholipid strips. The strips were incubated with 10 ng ml-1 of purified wild-type GST-Vps5 PX (left panel) or mutated GST-vps5Y322A,R360A PX domain fusion protein (right panel). PI, phosphatidylinositol; PC, phosphatidylcholine.

 

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