A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic - PubMed (original) (raw)

A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic

M Cormont et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The small GTPase Rab4 is implicated in endocytosis in all cell types, but also plays a specific role in some regulated processes. To better understand the role of Rab4 in regulation of vesicular trafficking, we searched for an effector(s) that specifically recognizes its GTP-bound form. We cloned a ubiquitous 69-kDa protein, Rabip4, that behaves as a Rab4 effector in the yeast two-hybrid system and in the mammalian cell. Rabip4 contains two coiled-coil domains and a FYVE-finger domain. When expressed in CHO cells, Rabip4 is present in early endosomes, because it is colocated with endogenous Early Endosome Antigen 1, although it is absent from Rab11-positive recycling endosomes and Rab-7 positive late endosomes. The coexpression of Rabip4 with active Rab4, but not with inactive Rab4, leads to an enlargement of early endosomes. It strongly increases the degree of colocalization of markers of sorting (Rab5) and recycling (Rab11) endosomes with Rab4. Furthermore, the expression of Rabip4 leads to the intracellular retention of a recycling molecule, the glucose transporter Glut 1. We propose that Rabip4, an effector of Rab4, controls early endosomal traffic possibly by activating a backward transport step from recycling to sorting endosomes.

PubMed Disclaimer

Figures

Figure 1

Obtention and characteristics of Rabip4 sequence. (A) Deduced amino acid sequence of the ORF in the Rabip4 cDNA. (B) Predicted structural organization of Rabip4 functional domains and tissue distribution of Rabip4 mRNA. The numbers at the top indicate the amino acid residues that define the boundaries of the coiled-coil (CC) and FYVE domains. The position of the I1 clone is indicated. The mouse tissue Northern blot was hybridized with the labeled probe corresponding to the 1.8-kb Rabip4 cDNA. H, heart; B, brain; S, spleen; Lu, lung; Li, Liver; SM, skeletal muscle; K, kidney; T, testis.

Figure 2

Confocal immunofluorescence and electron microscopy of CHO cells expressing GFP-Rabip4. CHO cells were transiently transfected with pEGFP Rabip4 or pEGFP Rabip4 Δ507–517 or stably transfected with pEGFP Rabip4 for the electron microscopy studies. Cells were treated for confocal analysis (Upper) or electron microscopy (Lower). GFP-fusion proteins were observed by using FITC parameters and the figures show 0.15–0.25 μm sections of CHO cells made by confocal analysis. Bars correspond to 1 μm (Upper), 250 nm (Lower), and 100 nm (Inset). Arrows point to vesicles 150–200 nm wide. The_Inset_ shows an immunolocalization using anti-Rabip4 serum and 10 nm colloidal gold-conjugated secondary antibody. Numerous gold beads labeled the vesicular structures, whereas no significant labeling was obtained on control cells or when anti-Rabip4 serum was omitted.

Figure 3

Rabip4 is localized in early sorting endosomes and gives enlarged vesicles with active Rab4. CHO cells were transiently transfected with pcDNA3-Rabip4 and pEGFP-Rab4 (a), pEGFP-Rabip4 (b and c), pEGFP-Rabip4 and pcDNA3 myc-Rab4 Q67L (d), or pEGFP-Rabip4 Δ(507–517) and pcDNA3 myc-Rab4 Q67L (e). Rabip4 is detected by using anti-Rabip4 antiserum and Texas Red-coupled anti-rabbit Ig (a) and myc-Rab4 Q67L is detected with mAb anti-myc followed by Texas Red-coupled anti-mouse Ig (d and_e_). Cells overexpressing GFP-Rabip4 were incubated with mAb anti-EEA1 (b) or with anti-Rab11 purified polyclonal Ig (c), followed by Texas Red-coupled anti-species Ig. Rab11 labeling was visible only in sections corresponding to the top of the cells (c), whereas no labeling was obtained when nonimmune Ig was used. The figures show merged images of green (GFP-labeled proteins) and red labeling obtained for the same section of representative CHO cells, with yellow color resulting from the overlay of green and red. (Bars = 1 μm.)

Figure 4

Rabip4 increases the overlap between sorting and recycling endosomes. Control CHO cells (Left) or cells transfected with pcDNA3 Rabip4 (Right) were cotransfected with pEGFP Rab5 and myc-Rab4 Q67L, with pEGFP Rab4 Q67L and myc-Rab11 or pEGFP Rab7 and myc Rab4 Q67L. Myc-tagged proteins and Rabip4 were detected as in Fig. 3. Figures represent the merged images of green and red labeling corresponding to active Rab4 compared with Rab5, Rab11, and Rab7. (Bar = 1 μm.)

Figure 5

Glut 1-myc distribution in cells expressing GFP-Rabip4. WT (a) or stably expressing GFP-Rabip4 (b_–_d) CHO cells were transiently transfected with pCis2 Glut 1-myc alone (a and_b_) or together with pCis2 Rab4 Q67L (c) or pCis2 Rab4 N121I (d). Three days after transfection, cells were fixed and permeabilized. Glut 1-myc is detected with anti-myc mAb and Texas red-coupled anti-mouse Ig. The merged images corresponding to green Rabip4 and red Glut 1-myc of the same confocal section are shown. Cells overexpressing Rab4 were identified by using anti-Rab4 serum and Cy5-coupled anti-rabbit Ig (data not shown). (Bar = 1 μm.)

References

1. Chavrier P, Goud B. Curr Opin Cell Biol. 1999;11:466–475. - PubMed
1. Morhmann K, van der Sluijs P. Mol Membr Biol. 1999;16:81–87. - PubMed
1. van der Sluijs P, Hull M, Webster P, Mâle P, Goud B, Mellman I. Cell. 1992;70:729–740. - PubMed
1. Trischler M, Soorvogel W, Ullrich O. J Cell Sci. 1999;112:4773–4783. - PubMed
1. Seachrist J L, Anborgh P H, Ferguson S G S. J Biol Chem. 2000;275:27221–27228. - PubMed

A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic - PubMed (original) (raw)