Amino acids 100, 101, 105-109 of Rabenosyn-5 are required for its interaction with hVps45. (A) The yeast strain AH109 was co-transformed with the following GAL4BD fusion constructs: GAL4BD-Vps45 and GAL4BD-p53 (control) together with the following GAL4AD fusion constructs: GAL4AD-R-5 50-200, GAL4AD-R-5 50-200 95-99 ala, GAL4AD-R-5 50-200 100, 101 ala, GAL4AD-R-5 50-200 102-104 ala, GAL4AD-R-5 50-200 105-109 ala, GAL4AD-R-5 50-200 110-114 ala, and GAL4AD-SV40 Large T-antigen (control). Co-transformants were assayed for their growth on non-selective (+HIS) and selective (−HIS) media. (B–D), HeLa cells were either grown on 35 mm dishes (B) or coverslips (C and D) and were co-transfected with either Myc-Vps45 and HA-R-5 or Myc-Vps45 and HA-R-5 100, 101, 105-109 ala. After 24 h, cells were harvested (B), lysed and in some cases, subjected to immunoprecipitations with antibody against the HA epitope. Lysates and immunoprecipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either anti-HA or anti-Myc antibodies followed by anti-mouse-HRP conjugated antibody. (C and D) transfected cells were fixed and immunostained with antibody against the HA epitope followed by 568-conjugated anti-mouse antibody. Bar, 10 μm.

Inhibition of proteasomal degradation stabilizes Rabenosyn-5 and hVps45 expression. HeLa cells were either grown on coverslips (A–D) or 35 mm dishes (E) and either Mock-treated (A, C, and E) or treated with hVps45-siRNA (B, D, and E). After 36 h, cells were either left untreated (control in A, B, and E) or subjected to treatment with 5 μM of the proteasomal inhibitor lactacystin (C, D, and E) for an additional 36 h. (A–D) Cells were fixed and immunostained with anti-Rabenosyn-5 antibody followed by 568-conjugated anti-rabbit antibody. (E) Cells were harvested, lysed, resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-hVps45 and anti-actin antibodies followed by anti-mouse HRP and anti-rabbit HRP conjugated antibodies (left panels), or trypsinized, fixed with 4% paraformaldyhide, immunostained with anti-Rabenosyn-5 antibody followed by 488-conjugated anti-rabbit antibody, and subjected to flow cytometry analysis (right panel). Bar, 10 μm.

hVps45 regulates cell migration. (A–E) Human foreskin fibroblast cells were grown on coverslips coated with fibronectin. Cells were either Mock-treated or treated with hVps45-siRNA for 48h. Scratches (wounds) approximately 850 μm wide were introduced onto the coverslips using a pipet tip. Fields of cells were fixed immediately after introducing the scratch (A, C), whereas the cells shown in B and D were fixed 6 h later. Cells were immunostained with 488-conjugated anti-wheat germ agglutinin antibody, which marked the outlines of the cells. (E) Quantification of three independent experiments as performed in A–D. Error bars display standard error and asterisk denotes p-values less than 0.05.

hVps45-depletion affects the subcellular distribution of both Syntaxin6 and Syntaxin16 and reduces Syntaxin16 protein levels. (A) Cells were either Mock-treated or treated with hVps45-siRNA for 72 h, harvested, lysed, resolved by 8% SDS-PAGE, immunoblotted with either anti-hVps45, anti-actin, anti-Syntaxin6 or anti-Syntaxin16 antibodies followed by appropriate HRP-conjugated antibodies. (B–M) Cells were grown on coverslips and either Mock-treated (B–D and H–J) or treated with hVps45-siRNA (E–G and K–M) for 72 h. Cells were then immunostained with either Syntaxin16 and GM130 antibodies (B–G) or Syntaxin6 and GM130 antibodies (H–M) followed by 568-conjugated goat anti-mouse and 488-conjugated goat anti-rabbit antibodies.

hVps45 interacts with Rabenosyn-5 in mammalian cells. (A) HeLa cells were harvested, lysed, and subjected to immunoprecipitations with either antibody against human Rabenosyn-5 (R-5) or pre-immune serum (Pre-Imm). Immunoprecipitates and lysates were resolved by 8% SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with either anti-R-5 (left panel), anti-R-5 or anti-hVps45 (center panel; nitrocellulose was cut and blotted with either antibody as indicated by the border line), and with either anti-FAK or anti-Hsc70 (far right panel; nitrocellulose was cut and blotted with either antibody as indicated by the border line) and followed by secondary anti-rabbit HRP-conjugated antibody. (B) The yeast strain AH109 was co-transformed with the following GAL4 binding domain (GAL4BD) fusion constructs: GAL4BD-Vps45 and GAL4BD-p53 (control) together with the following GAL4 transcription activation domain (GAL4AD) fusion constructs: GAL4AD-R-5 (full length), GAL4AD-R-5 1-623, GAL4AD-R-5 1-263, GAL4AD-R-5 50-200, GAL4AD-R-5 70-170, GAL4AD-R-5 70-152, GAL4AD-R-5 70-120, GAL4AD-R-5 130-170, GAL4AD-R-5 100-170, and GAL4AD-SV40 Large T-antigen (control). Co-transformants were assayed for their growth on non-selective (+HIS) and selective (−HIS) media. (C) HeLa cells were co-transfected with either Myc-Vps45 and HA-R-5 or Myc-Vps45 and HA-R-5 264-784. After 24 h, cells were harvested, lysed, and in some cases, subjected to immunoprecipitations with antibody against the HA epitope. Lysates and immunoprecipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either anti-HA or anti-Myc antibodies followed by anti-mouse-HRP conjugated antibody. IP=immunoprecipitation; IB=immunoblot; Ig=immunoglobulin; C2H2=zinc finger domain; FYVE=PtdIns3P-binding domain; CC=coiled-coil region; NPF=asparagine-proline-phenylalanine motif.

Decreased expression of Rabenosyn-5 upon hVps45-depletion. (A) HeLa cells were either Mock-treated, treated with scrambled-siRNA, or treated with hVps45-siRNA for 72 h. Cells were harvested and lysates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-hVps45, anti-Rabenosyn-5, and anti-actin antibodies followed by anti-mouse-HRP conjugated and anti-rabbit-HRP conjugated antibodies. (B) Cells were grown on coverslips and were either Mock-treated (upper panels) or treated with hVps45-siRNA (lower panels) for 72 h. Cells were then fixed and immunostained with anti-Rabenosyn-5 antibody followed by 568 conjugated anti-mouse antibody and DAPI. (C) Cells were either Mock-treated or treated with hVps45-siRNA. After 48 h, cells were left untreated or subjected to the lysosomal inhibitors NH₄Cl or leupeptin for 24 h. Cells were harvested, lysed, resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Rabenosyn-5, anti-LDL receptor (LDL-R, control), anti-hVps45, and anti-actin (control) antibodies followed by anti-mouse-HRP and anti-rabbit-HRP conjugated antibodies. Bar, 10 μm.

hVps45 and Rabenosyn-5 regulate β1 integrin recycling. HeLa cells were grown on coverslips and either Mock-treated (A and B), treated with hVps45-siRNA (C and D), or treated with Rabenosyn-5-siRNA (E and F) for 48 h. Cells were incubated in starvation media (DMEM + 0.5% BSA) for 1 h, “pulsed” with anti-β1 integrin antibody in starvation media for 1 h at 37°C (A, C, and E), and “chased” in complete media (DMEM + 10% fetal calf serum) for 2 h, and then acid-stripped to remove β1 integrin that had returned to the plasma membrane (B, D, and F). After fixation and permeabilization, the cells were immunostained with 568-conjugated anti-mouse antibody. (G) Quantification of the experiments performed in A–F. Images of 150 cells from each treatment were taken using a Zeiss LSM5 PASCAL confocal microscope, and the mean intracellular signal intensity of non-recycled β1 integrin was analyzed by using LSM5 PASCAL software. (H) Cells were grown on coverslips and either Mock-treated or treated with hVps45-siRNA. After 6 h, the cells were transfected with a GFP-Vps45 construct resistant to hVps45-siRNA oligonucleotides. 66 h later, cells were incubated in starvation media for 1 h, “pulsed” with β1 integrin antibody in starvation media for 1 h at 37°C, “chased” in complete media for 3 h, stripped, fixed, and immunostained with 568 conjugated anti-mouse antibody. Images of cells from each treatment (180 cells in H) were analyzed. (I) Cells were grown on coverslips and either Mock-treated or treated with Rabenosyn-5-siRNA. After 24 h, cells were transfected with either siRNA-resistant GFP-Rabenosyn-5 (si-GFP-R-5) or the siRNA-resistant Rabenosyn-5 mutant that is incapable of Vps45-binding (si-GFP-R-5 100, 101, 104-109 ala), for an additional 48 h. Then, cells were subjected to β1 integrin recycling assay as described in H and images of cells from each treatment (150 cells) were analyzed. Error bars display standard error and asterisks denote p-values less than 0.01 utilizing the t-Test for independent samples (in G). Results were derived from 3 independent experiments. Bar, 10 μm.

Depletion of hVps45 affects retromer localization. HeLa cells were grown on coverslips and were Mock-treated or treated with hVps45-siRNA for 72 hours prior to fixation. Cells were then co-immunostained with anti-SNX1 and anti-Giantin (A–F and M), and with anti-hVps35 and anti-GM130 (G–L and N) followed by 568 conjugated anti-mouse and 488 conjugated anti-rabbit or 488 conjugated anti-goat antibodies. (M and N) Co-localization between SNX1 and Giantin (M), and between hVps35 and GM130 (N), respectively, were quantified using ImageJ software. Error bars display standard error and asterisks denote p-values less than 0.01 utilizing the t-Test for independent samples. Results were derived from 3 independent experiments by counting 100 cells from each experiment. Insets (F and L) depict the co-staining in a single cell. Bar, 10 μm.