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First published online March 8, 2006
doi: 10.1242/10.1242/jcs.02810

Journal of Cell Science 119, 1053-1062 (2006)
Published by The Company of Biologists 2006
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Varp is a Rab21 guanine nucleotide exchange factor and regulates endosome dynamics

Xinjun Zhang1,2, Xi He3, Xin-Yuan Fu1,2,4,* and Zhijie Chang1,2,*

1 Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing (100084), China
2 Department of Institute of Biomedicine, Tsinghua University, Beijing (100084), China
3 Division of Neuroscience, Children's Hospital, Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, USA


Figure 1
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  		Fig. 1. The Varp protein sequence. (A) Alignment of human Rabex5 and Varp N-terminal fragment. The solid line indicates the VPS9 domain. (B) Alignment of a part of the VPS9 domains from human Rabex5 (hRabex5), human RIN1 (hRIN1), human ALS2 (hALS2), S. cerevisiae Vps9p (scVps9), human Varp (hVarp) and mouse Varp (mVarp) protein sequences. The shaded regions (black and gray) represent identical and conserved amino acids, respectively. Arrowheads indicate the four crucial amino acids in Rabex5 required for the GEF activity.

 

Figure 2
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  		Fig. 2. Expression of Varp mRNA during (A) mouse embryogenesis, in (B) adult mouse tissues and in (C) human cell lines. A pair of specific primers for the mouse or human Varp gene was used for the RT-PCR analysis; ß-actin was used as an internal control.

 

Figure 3
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  		Fig. 3. The VPS9 domain of Varp catalyzes Rab21 guanine nucleotide exchange reaction in vitro. (A) A schematic diagram of the full-length Varp and VarpN protein. VPS9, vacuolar protein sorting 9 domain; ANK, ankyrin repeat. (B) Flag-tagged Varp and VarpN proteins purified from transfected HEK293T cells were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. (C) [3H]GDP-loaded His6-Rab5a was incubated with the purified Flag-tagged Varp, VarpN and Rabex5. The percentage of [3H]GDP remained bound to Rab5a after 60 minutes is presented. Each value represents the mean and standard deviation of at least two independent assays. (D) His6-Rab5a was incubated with [{gamma}-35S]GTP at 30°C in the presence of Flag-tagged Varp, VarpN or Rabex5 for 60 minutes. Counts of the bound [{gamma}-35S]GTP on Rab5a are presented. Each value represents the mean and standard deviation of at least two independent assays. (E) A total of six Rab GTPases, including Rab4, Rab5a, Rab7, Rab15, Rab21 and Rab22, were subjected to the in vitro [{gamma}-35S]GTP-binding assay. Each GST-fused Rab GTPase was incubated with [{gamma}-35S]GTP at 30°C in the presence or absence of Flag-tagged VarpN for 60 minutes. Each value represents the mean and standard deviation of at least two independent assays. Empty, no Rab protein.

 

Figure 4
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  		Fig. 4. Varp interacts with Rab21 and Rab5a. Coimmunoprecipitation of the full-length Varp protein and Rab5a or Rab21, or their mutant forms. Myc-tagged Varp (Varp-Myc) and each of the GFP-tagged Rabs were coexpressed in HEK293T cells. Whole-cell lysates were immunoprecipitated with an anti-Myc antibody and precipitates were analyzed by western blotting with an anti-GFP antibody. Aliquots of whole-cell lysates were also examined by western blotting with anti-GFP or anti-Myc antibodies.

 

Figure 5
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  		Fig. 5. Ectopically expressed Varp colocalizes with endosomal markers. (A) HeLa cells stably expressing Varp-Myc were used in these assays. (a-c) Cells were fixed and double labeled with a rabbit polyclonal anti-Varp antibody (green) and a mouse anti-EEA1 antibody (red). (d-f) Cells were transfected with an EGFP-Rab5a construct and labeled with anti-Myc antibody (red). (g-h) Cells were labeled with 25 µg/ml Alexa 594-conjugated human transferrin in DMEM at 37°C for 10 minutes and washed with ice-cold PBS, and then fixed and immunostained with an anti-Varp antibody (green). (B) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab5a construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate the colocalization of Varp, EEA1 and Rab5. (C) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab21 construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate the colocalization of Varp, EEA1 and Rab21. (D) Control HeLa cells or HeLa cells expressing the Varp protein were transfected with an EGFP-Rab7 construct and labeled with an anti-Varp antibody (red) and an anti-EEA1 antibody (blue). Arrows indicate colocalization of Varp, EEA1 and Rab7, and arrowheads indicate colocalization of Varp and Rab7, but not EEA1. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Ectopic expression of Varp induces giant vacuoles in HeLa cells. (A) Lysates from HeLa cells stably expressing Myc-tagged Varp, VarpN, or Rabex5 were separated by SDS-PAGE and immunoblotted (IB) with an anti-Myc antibody. (B) Phase-contrast micrographs of living HeLa cells stably expressing Varp (a), VarpN (b), Rabex5 (c), or transiently expressing EGFP-Rab5Q79L (d). Bars, 50 µm. (C) Late endosomal and/or lysosomal markers are localized on the giant vacuoles in HeLa cells expressing the Varp protein. HeLa cells expressing Varp were labeled with (a-c) anti-Varp (green) and anti-EEA1 antibodies (red), or (g-i) anti-LAMP3 antibody (red). (d-f) HeLa cells expressing Varp were transfected with an EGFP-Rab7 construct and labeled with an anti-Varp antibody (red). (j-l) HeLa cells expressing EGFP-tagged Varp were labeled with a rabbit anti-p115 antibody (red). Bars, 10 µm. (D) HeLa cells expressing Varp were transfected with an EGFP-Rab21 construct and labeled with an anti-Varp antibody (red). Colocalization of Varp with EGFP-Rab7, LAMP3 or EGFP-Rab21 on the giant endosomes is also shown (arrows). *, giant endosomes induced by Varp. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Varp does not associate with Rabaptin-5. Flag-tagged Rabaptin-5 (Flag-Rabaptin-5) and Myc-tagged Varp (Varp-Myc), or Myc-tagged Rabex5 were coexpressed in HEK293T cells. Whole-cell lysates were immunoprecipitated with an anti-Myc antibody and precipitates were analyzed by western blotting with an anti-Flag M2 antibody. Aliquots of whole-cell lysates were also examined by western blotting with anti-Flag or anti-Myc antibodies.

 

Figure 8
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  		Fig. 8. Wortmannin disrupts the association of Varp protein with the early endosomes. HeLa cells expressing Varp were treated with DMSO or 100 nM wortmannin for 30 minutes at 37°C. The cells were fixed and stained with anti-Varp (red) and anti-EEA1 (green) antibodies. Bars, 10 µm.

 

Figure 9
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  		Fig. 9. The activation and endosomal localization of Rab21 depends on the endogenous Varp protein. (A) HeLa cells were transfected with a Varp-siRNA- or control siRNA-expressing construct and the cell lysates were analyzed by western blotting with anti-Varp and anti-Hsp70 antibodies 72 hours post transfection. (B) HeLa cells were transfected with (a-f) indicated siRNA- and EGFP-Rab21 WT-expressing plasmids for 72 hours, and were fixed and labeled with an anti-EEA1 antibody (red). (g-i) HeLa cells expressing EGFP-Rab21 T33N were labeled with an anti-EEA1 antibody. Bars, 10 µm.

 

Figure 10
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  		Fig. 10. The VPS9 domain and ankyrin repeats are required for the subcellular localization and activity of Varp in HeLa cells. (A) Schematic diagram of the Varp protein and its mutants used in this experiment. (B) Cell lysates from cells expressing EGFP-fused Varp proteins were analyzed by western blotting with anti-GFP and anti-Varp antibodies. (C) Distribution of Varp and its mutants. HeLa cells ectopically expressing EGFP-fused proteins were fixed and labeled with an anti-EEA1 antibody (red); (c,f,i,l,o) show the co-staining of Varp or mutants with the early endosomal marker EEA1. The effect of the full-length or Varp mutants on the size of early endosomes was also analyzed (b,e,h,k,n). Bars, 10 µm. Arrows indicate the colocalization of Varp and EEA1 on the enlarged early endosomes; *, giant late endosome. (D) Colocalization of Varp/{Delta}VPS and late endosomal markers. (a-c) HeLa cells transfected with Myc-tagged Varp/{Delta}VPS and EGFP-tagged Rab5 were stained with an anti-Myc antibody (red). (d-f) HeLa cells transfected with Myc-tagged Varp/{Delta}VPS and EGFP-tagged Rab7 were stained with anti-Myc antibody (red). (g-i) HeLa cells expressing EGFP-tagged Varp/{Delta}VPS were stained with an anti-LAMP3 antibody (red). Bars, 10 µm.

 

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© The Company of Biologists Ltd 2006