Role of HRB in clathrin-dependent endocytosis

Abstract

Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.

FIGURE 1.

Interaction of HRB and TI-VAMP. A, HRB was identified as a new partner of TI-VAMP in three independent yeast two-hybrid screens. Results of yeast two-hybrid screens using either the Longin domain (amino acids 1-120) or the cytoplasmic domain of TI-VAMP (amino acids 1-188) and either a human placenta or a human fetal brain cDNA library are shown. HRB was found in the three different screens. Green lines highlight the total coverage of all prey clones identified in the screens, and red lines represent the intersection of the clones. Relevance of each identified interaction was noted as A on an A to E predicted biological score scale, showing a very high confidence on the interaction. The interaction with TI-VAMP is mediated by a minimal interaction domain of 70 amino acids (amino acids 147-217) of HRB. B, Western blot analysis of the expression of HRB in different cell lines and in rat brain. A 60-kDa band corresponding to HRB is observed in different cell types and in rat brain. Two bands of different apparent molecular mass that could correspond to different isoforms of the protein are observed in brain during development. C, in vitro interaction of HRB and TI-VAMP. Glutathione beads coated with GST or GST-TI-VAMP (1 μm) were incubated overnight with in vitro translated human HRB in buffer containing Triton X-100 (Tx100) or Nonidet P-40 (NP40) as detergent. The fractions bound to glutathione beads were eluted and separated by SDS-PAGE, and HRB was detected by Western blot. More in vitro translated HRB is bound to GST-TI-VAMP beads than to GST beads, indicating an interaction between HRB and TI-VAMP. D, in vitro interaction of HRB and the Longin domain of TI-VAMP. GST pulldown experiments were performed with recombinant GST, GST-TI-VAMP, and GST-Longin and with total rat brain extracts. HRB bound to the cytoplasmic domain and to the Longin domain of TI-VAMP. E, HRB interacts with TI-VAMP in the brain. Five P4 rat brains were homogenized, and the Triton X-100-soluble fraction was immunoprecipitated with a monoclonal antibody against TI-VAMP or with mouse IgG as a negative control. Co-immunoprecipitated endogenous HRB (arrowhead) and IgG (asterisk) were detected by Western blot. aa, amino acids; E16, embryonic day 16; NGF, nerve growth factor; TIV, TI-VAMP; mIgG, mouse IgG; WB, Western blot; IP, immunoprecipitation; Ad., adult; SM, starting material; Arf-GAP, ADP-ribosylation factor GTPase-activating protein.

FIGURE 2.

The subcellular localization of HRB is compatible with a role in endocytosis. HeLa cells were fixed and stained for endogenous HRB (in green) and the indicated markers of the clathrin-dependent endocytic pathway (in red). Arrowheads point to structures labeled by both HRB and the indicated marker (bar, 10 μm). Note the high degree of colocalization of HRB with AP-2 and EPS15. HRB also partially colocalizes with clathrin light chain and transferrin receptor, indicating that HRB likely localized in clathrin-coated structures. HRB was also present in a diffuse manner in the nucleus.

FIGURE 3.

Localization of TI-VAMP and HRB in HeLa and MDCK cells. A, HeLa cells were fixed and stained for endogenous HRB (in green) and TI-VAMP (in red). Arrowheads point to structures positive for only one of the proteins. No significant colocalization was observed at steady state, indicating that HRB and TI-VAMP could interact transiently (bar, 10 μm). B, MDCK cells expressing inducible GFP-TI-VAMP were fixed and stained for the endogenous HRB (in red). Arrowheads point to structures labeled by both HRB and GFP-TI-VAMP (bar, 10 μm).

FIGURE 4.

HRB colocalizes with endocytosed TI-VAMP. HeLa cells were transfected with pHLuorin-TI-VAMP and then incubated with a GFP antibody at 37 °C to follow the endocytosis of pHLuorin-TI-VAMP. Cells were washed, fixed at the indicated time points, and stained for endogenous HRB (acquired in Cy5 channel, green) and endocytosed anti-GFP antibody (red). Arrowheads point to structure where HRB and pHLuorin-TI-VAMP are both present. Note the good degree of colocalization after 5 min of endocytosis suggesting that the interaction between both proteins may be restricted to the early stages of endocytosis (bar, 10 μm). mGFP, mouse monoclonal anti-GFP antibody.

FIGURE 5.

siRNA- and shRNA-mediated gene silencing of HRB in HeLa cells. A, cells were transfected by siRNA against HRB or luciferase (Luc) as a negative control, lysed, and analyzed by Western blot 24 h later. HRB protein is detected at 60 kDa. Extinction is about 35% with siRNA against HRB. B, stable HeLa cell lines were established using a control shRNA targeting no known human gene targets (Ctrl) or two different shRNAs directed against two different regions of the human HRB coding sequence (shRNA 1 and shRNA 2). Cells were lysed, and HRB was detected by Western blot. Extinction of HRB is around 90 and 50% for shRNA 1 and shRNA 2, respectively. Bottom panel, control (Ctrl) and HRB knockdown cells (shRNA 1) were fixed and stained for endogenous HRB (bar, 10 μm). Some HRB staining still remains in the nucleus but is virtually absent from the cytoplasm of shRNA 1 cells. RNAi, RNA interference. ^**, p < 0.01; ^***, p < 0.001 in Student's t test. Error bars represent S.E. throughout.

FIGURE 6.

Inhibition of the expression of HRB has no effect on the localization of endogenous TI-VAMP. Control (Ctrl) and HRB knockdown cells (shRNA 1) cells were fixed and stained for endogenous HRB and TI-VAMP (bar, 10 μm). Note that endogenous TI-VAMP shows the same perinuclear pattern in both cases.

FIGURE 7.

Inhibition of the expression of HRB inhibits transferrin and TI-VAMP endocytosis. A, control (Ctrl) or HRB knockdown cells (shRNA 1 and shRNA 2) were harvested and incubated with fluorescent Tf-Alexa 488 (A488) for 5, 15, or 30 min at 37 °C. Cells were then washed, and surface labeling was stripped with acid buffer. Endocytosed Tf was quantified by fluorescence-activated cell sorting. Each time point corresponds to 20,000 cells. The y axis represents arbitrary units of fluorescent Tf. HRB knockdown cells showed a significant inhibition of Tf endocytosis (Student's t test: control/shRNA 1, p = 1.8·10^-6, 4·10^-6, and 3.5·10^-6 at 5, 15, and 30 min, respectively; control/shRNA 2, p = 2.8·10^-4, 2.8·10^-4, and 8.2·10^-3 at 5, 15, and 30 min, respectively. Right panel, detailed analysis of one representative flow cytometry experiment measuring Tf endocytosis in control and HRB knockdown cells (shRNA 1) after 30 min of endocytosis. Control and HRB knockdown cells (shRNA 1) showed a significant increase in Tf median fluorescence compared with the unlabeled cells (corresponding to 0 min of endocytosis). HRB knockdown cells (shRNA 1) showed a significant decrease in Tf median fluorescence compared with the control cells (shown here by a left shift of the fluorescence intensity). B, HeLa cells were transfected with siRNA against HRB or luciferase. After 24 h, cells were starved and incubated at 37 °C with Tf-Alexa 488 for 30 min. Cells were then fixed and processed for immunofluorescence. HRB knockdown cells accumulate less fluorescent Tf than mock cells (treated with luciferase siRNA). C, control or HRB stably depleted cells (shRNA 1) were transfected with pHLuorin-TI-VAMP construct, harvested, and incubated with an anti-GFP antibody at 4 °C to stain surface TI-VAMP. Cells were then incubated at 37 °C for 20 min to allow endocytosis, extensively washed, fixed, and incubated with an anti-mouse antibody coupled to Cy5 to stain the pool of TI-VAMP that remains in the surface of the cells. This pool was quantified by fluorescence-activated cell sorting. Each measure corresponds to 20,000 cells. The y axis represents the percentage of surface-bound pHLuorin-TI-VAMP endocytosed in 20 min. HRB knockdown cells showed a significant decrease of ∼50% of TI-VAMP endocytosis (Student's t test: control/shRNA 1, p = 0.028). ^*, p < 0.05; ^***, p < 0.001 in Student's t test. mAb, monoclonal antibody.