TIP47 is a key effector for Rab9 localization

Abstract

The human genome encodes approximately 70 Rab GTPases that localize to the surfaces of distinct membrane compartments. To investigate the mechanism of Rab localization, chimeras containing heterologous Rab hypervariable domains were generated, and their ability to bind seven Rab effectors was quantified. Two chimeras could bind effectors for two distinctly localized Rabs; a Rab5/9 hybrid bound both Rab5 and Rab9 effectors, and a Rab1/9 hybrid bound to certain Rab1 and Rab9 effectors. These unusual chimeras permitted a test of the importance of effector binding for Rab localization. In both cases, changing the cellular concentration of a key Rab9 effector, which is called tail-interacting protein of 47 kD, moved a fraction of the proteins from their parental Rab localization to that of Rab9. Thus, relative concentrations of certain competing effectors could determine a chimera's localization. These data confirm the importance of effector interactions for Rab9 localization, and support a model in which effector proteins rely on Rabs as much as Rabs rely on effectors to achieve their correct steady state localizations.

Figure 1.

Rab chimeras studied. (A) Ribbon diagrams of the Rab5c-GppNHp (residues 19–182, left; Merithew et al., 2001) and Rab9-GDP (residues 2–175, right; Chen et al., 2004) structures (Pettersen et al., 2004). The so-called complementarity determining regions that are believed to be important for effector interaction (Ostermeier and Brunger, 1999) are highlighted in red. The first conserved basic residue that was used as the site of hypervariable domain exchange is highlighted in blue; the presumed Rab switch regions (Ostermeier and Brunger, 1999; Merithew et al., 2001) are highlighted in yellow. (B) Diagrams of the hypervariable domain chimeras described herein. Residue numbers indicate composition of the constructs; Rabs differ in length at both NH₂ and COOH termini, so the junction numbers differ and are shown to represent the precise junction identities from the parental Rab sequences.

Figure 2.

Chimeric Rabs are prenylated and active for GTP binding. (A and B) Analysis of GFP-Rab membrane association in HeLa cells. HeLa cells were transiently transfected for GFP-Rab expression, and lysates were separated into membrane and cytosol fractions for analysis by SDS-PAGE and anti-GFP immunoblot. Bands shown in A were quantitated by ImageJ for display in B. (C) Nucleotide (GTPγS) binding to recombinant, purified GST-Rabs. Rabs were incubated with radiolabeled GTPγS, and the extent of binding was quantitated by nitrocellulose filter binding and scintillation counting. Error bars represent the SEM.

Figure 3.

The Rab9 hypervariable domain is necessary and sufficient for Rab9 effector binding. (A) Binding of purified, untagged Rab9, Rab1A, Rab9/1, and Rab1/9 to the Rab9 effectors His₆-TIP47(152–434; closed bars) and His₆-p40 (open bars). Data are normalized relative to the binding seen for parental Rabs. (B) Same as in A, with untagged Rab9, Rab5a, Rab9/5, and Rab5/9. (C) Binding of [³⁵S]GTPγS- and [³H]GDP-loaded Rab9, Rab5/9, and Rab1/9 to His₆-TIP47(152–434). 100% binding to p40 or TIP47 represented 3.9 or 1.5 pmol Rab9, respectively, in A and B. This assay measures active Rab molecules only; untagged Rab proteins were all ∼100% active, except for Rab9/5, which contained ∼30% active molecules. Error bars represent the SEM.

Figure 4.

Some, but not all, Rab1 and Rab5 effectors require hypervariable domain sequences for binding. (A) Binding of GST versions of Rab9, Rab5, Rab9/5, and Rab5/9 to Rab5 effectors EEA1 and rabaptin-5 from cytosol. (B) Binding of GST versions of Rab9, Rab1, Rab9/1, and Rab1/9 to Rab1 effectors GM130, golgin-84, and p115 from rat liver Golgi detergent extracts. Data are normalized relative to the binding seen for parental Rabs. Error bars represent the SEM.

Figure 5.

Colocalization of Rab5/9 with EEA1. (A) Colocalization of CFP-Rab5/9 with the early endosome marker EEA1 in fixed HeLa cells stained with anti-EEA1 and anti-GFP antibodies. The square in A is enlarged for both markers (right); arrows are included to facilitate comparison. Bar, 10 μm. (B) Quantitation of CFP-Rab5/9, CFP-Rab5, and YFP-Rab9 colocalization. Venn diagrams are shown as follows: CFP-Rab5/9, CFP-Rab5, and YFP-Rab9 versus EEA1; CFP-Rab5/9 and CFP-Rab5 versus endocytosed anti–CI-MPR IgG; and CFP-Rab5/9 and CFP-Rab5 versus YFP-Rab5. The number of positive structures is indicated below each protein name; overlap is indicated as described in the Results section.

Figure 6.

TIP47 binding to Rab5/9 triggers relocalization from early to late endosomes. (A) Wide-field light microscopy. GFP-Rab5/9 localization in fixed HeLa cells (left). The overexpression of wild-type TIP47 (middle), but not of mutant TIP47^SVV-AAA (Hanna et al., 2002), which is deficient for Rab9 binding (right), causes GFP-Rab5/9 localization to perinuclear late endosomes. (middle) The cell is outlined in white. Levels of TIP47 wild-type and mutant protein overexpression were approximately ninefold. (B) TIP47-relocalized perinuclear Rab5/9 is present on late endosomes. Deconvolution microscopy of cells overexpressing TIP47 protein; perinuclear GFP-Rab5/9 colocalized with late endosomal, endocytosed anti–CI-MPR antibody. (C) Deconvolution microscopy shows TIP47- relocalized perinuclear Rab5/9 is not present on the Golgi, as it did not colocalize with the Golgi marker p115. In the merge images in B and C, Rab5/9 is shown in green, whereas the organelle markers are shown in red. In B and C, the TIP47 is shown as the mean of the summation of total Z-sections; other images are 0.2-μm Z-sections. Bars: (A) 10 μm; (B and C) 5 μm.

Figure 7.

TIP47 binding to Rab1/9 triggers relocalization from the Golgi to late endosomes. (A and B) Deconvolution microscopy shows CFP-Rab1/9 (left) and endocytosed anti–CI-MPR antibody (middle) localizations in HeLa cells in the presence of overexpressed wild-type (A) or Rab-binding–deficient mutant (B) TIP47 (right). The bottom images show the perinuclear region of the same cell. (C) Deconvolution microscopy shows CFP-Rab1/9 (left) and p115 (right) localizations in HeLa cells in the presence of overexpressed wild-type TIP47 (right). In the merge images, Rab1/9 is shown in green, whereas the organelle markers are shown in red. TIP47 is shown as the mean of the summation of total Z-section images; other images are 0.2-μm Z-sections. Bars, 5 μm.

Figure 8.

Model for how effectors contribute to membrane localization of Rab proteins. GDP-bound Rabs are recruited to membranes from cytosolic complexes with GDI through the catalytic action of a GDF. A GEF catalyzes nucleotide exchange. Interaction of the Rab-GTP with an effector that has been recruited to the same membrane by a Rab (option A) or by an effector-binding partner (option B) will stabilize both the Rab and the effector on the membrane. If not bound to the effector, a GTPase-activating protein (GAP) can activate the Rab to hydrolyze its GTP to GDP, thus, allowing for potential membrane extraction of the Rab by GDI. Thus, the Rab GTPase is dependent on its effectors, and vice versa, for stable interaction with the membrane. The effector-binding partner need not be a protein and can be particular phospholipids, such as phosphoinositides, that are specific to a given compartment (Zerial and McBride, 2001).