Functional cross-talk between Rab14 and Rab4 through a dual effector, RUFY1/Rabip4

Abstract

The small GTPase Rab14 localizes to early endosomes and the trans-Golgi network, but its cellular functions on endosomes and its functional relationship with other endosomal Rab proteins are poorly understood. Here, we report that Rab14 binds in a GTP-dependent manner to RUFY1/Rabip4, which had been originally identified as a Rab4 effector. Rab14 colocalizes well with Rab4 on peripheral endosomes. Depletion of Rab14, but not Rab4, causes dissociation of RUFY1 from endosomal membranes. Coexpression of RUFY1 with either Rab14 or Rab4 induces clustering and enlargement of endosomes, whereas a RUFY1 mutant lacking the Rab4-binding region does not induce a significant morphological change in the endosomal structures even when coexpressed with Rab14 or Rab4. These findings suggest that Rab14 and Rab4 act sequentially, together with RUFY1; Rab14 is required for recruitment of RUFY1 onto endosomal membranes, and subsequent RUFY1 interaction with Rab4 may allow endosomal tethering and fusion. Depletion of Rab14 or RUFY1, as well as Rab4, inhibits efficient recycling of endocytosed transferrin, suggesting that Rab14 and Rab4 regulate endosomal functions through cooperative interactions with their dual effector, RUFY1.

Figure 1.

Interaction of RUFY1 with Rab14 or Rab4. Yeast (AH109) transformants expressing the indicated combination of constructs were streaked (A) or spotted (B) onto plates lacking leucine and tryptophan, with or without histidine and adenine (+His, +Ade or −His, −Ade). (C and D) Pulldown assays for interaction of RUFY1 with Rab proteins. Lysates from HeLa cells transiently transfected with the EGFP-RUFY1 construct indicated were pulled down with the indicated GST-fusion protein. Bound proteins were subjected to SDS-PAGE and analyzed by immunoblotting using anti-GFP antibody. (E) HeLa cells expressing EGFP-tagged RUFY1 and either mCherry-tagged Rab14(Q70L) or mCherry were lysed and immunoprecipitated with anti-RFP antibody. Bound materials were subjected to SDS-PAGE and analyzed by immunoblotting using anti-RFP or anti-GFP antibody. (F) HeLa cells expressing HA-tagged Rab14(WT), Rab14(Q70L), Rab4(WT), or Rab4(Q67L) were fixed with methanol and doubly stained with anti-RUFY1 and anti-HA (3F10) antibodies. RUN domain (for RPIP8, UNC-14, and NESCA); cc, coiled-coil region; FYVE domain (for Fab1p, YOTB, Vac1p, and EEA1). Bar, 20 μm.

Figure 2.

Overlap of endosomes positive for Rab14 and Rab4. (A) HeLa cells were transiently cotransfected with HA-Rab14 and either EGFP-Rab4, EGFP-Rab5, EGFP-Rab7, or EGFP-Rab11, and processed for immunofluorescence microscopy. Arrows indicate endosomes with overlapping staining for Rab14 and another Rab protein. Green arrowheads, Rab5-, Rab7-, or Rab11-positive endosomes; and red arrowheads, Rab14-positive endosomes. To quantify colocalization, peripheral Rab14-positive endosomes (B) or peripheral endosomes positive for the Rab protein indicated (C) were used as reference. The quantification is based on two independent experiments, using 200–400 endosomes in 6–10 transfected cells. Bar, 20 μm.

Figure 3.

Depletion of Rab14, but not Rab4, abolishes endosomal localization of RUFY1. HeLa cells were treated with a pool of siRNAs for LacZ, Rab14 or both Rab4a and Rab4b. After 72 h, the cells were lysed to be processed for immunoblot analysis using antibodies to indicated proteins (A) or fixed with 3% PFA, permeabilized, and immunostained for Rab14 (B). The siRNA-treated cells were permeabilized with saponin before fixation with PFA and immunostained for Rab14 and RUFY1 using rabbit polyclonal anti-Rab14 and mouse polyclonal anti-RUFY1 antibodies. (C) The number of cells in which RUFY1 localized to endosomes was counted and normalized with total counted cell numbers. The graph is a representative of four independent experiments. (D) HeLa cells treated with siRNAs for Rab14 were transfected with a plasmid encoding HA-Rab14, fixed, and immunostained with anti-RUFY1 and anti-HA antibodies. Bars, 20 μm.

Figure 4.

Coexpression of RUFY1 and either Rab14 or Rab4 induces expansion of endosomes. (A) HeLa cells were transfected with a plasmid encoding HA-RUFY1, EGFP-Rab14, EGFP-Rab4, or EGFP-Rab5. In (a), the cells were stained with antibody to HA. (B) HeLa cells were cotransfected with plasmids encoding HA-RUFY1 and either EGFP-Rab14 (a), EGFP-Rab4 (b), or EGFP-Rab5 (c), and stained with anti-HA antibody. Bars, 20 μm.

Figure 5.

The enlarged endosomes are derived from early endosomes. HeLa cells cotransfected with plasmids encoding EGFP-RUFY1 and HA-Rab14 (A) or HA-Rab4 (B) were stained with anti-EEA1, anti-TfnR, or anti-Lamp-1 antibody. *, transfected cells; +, nontransfected cells. Bars, 20 μm.

Figure 6.

Deconvolution analysis of the enlarged endosomes. HeLa cells were cotransfected with plasmids encoding EGFP-RUFY1 and HA-Rab14 (A) or HA-Rab4 (B) and doubly stained with anti-HA and anti-EEA1 antibodies. (Ab, Bb and Bd) Deconvolved images of the boxed areas in Aa and Ba (two different areas, * and +, were analyzed) using Carl Zeiss AxioVision software. (Ac, Bc and Be) Digital magnification of the boxed areas in Ab, Bb and Bd, respectively. Bar, 20 μm.

Figure 7.

Coexpression of RUFY1(Δ507-517) and either Rab14 or Rab4 does not induce expansion of early endosomes. (A) HeLa cells transfected with a plasmid encoding EGFP-RUFY1 or EGFP-RUFY1(Δ507-517) were stained with anti-EEA1 antibody. (B and C) HeLa cells were cotransfected with a combination of plasmids encoding either EGFP-RUFY1 or EGFP-RUFY1(Δ507-517) and either HA-Rab14 (B) or HA-Rab4 (C), and stained with anti-HA antibody. Bar, 20 μm.

Figure 8.

Rab14 and RUFY1 are required for the efficient Tfn recycling. (A) HeLa cells treated with a pool of siRNAs for LacZ, Rab14, both Rab4a and Rab4b, or RUFY1 were incubated with AlexaFluor488-conjugated Tfn at 37°C for 5 min to internalize Tfn, acid-washed on ice to remove surface-bound Tfn, and further incubated at 37°C for indicated times to allow its transport and recycling to the cell surface. Bar, 20 μm. (B) Pixel intensity of AlexaFluor488-conjugated Tfn was estimated at the indicated time. The values are the mean ± SD of 100–200 cells at 0 min, 300–500 cells at 10 min, and >50 cells at 30 min. The graph is a representative of three independent experiments. Numbers indicate fold increase of the Tfn intensity at the 10-min time point.