Identification of residues in TIP47 essential for Rab9 binding

Abstract

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-dependent and cation-independent mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the trans-Golgi network in vitro and in living cells. TIP47 recognizes distinct determinants in the cytoplasmic domains of these two receptors, and its ability to bind to the cation-independent MPR is enhanced by the concomitant binding of the Rab9 GTPase. We show here that TIP47 residues 161-169 are essential, but likely not sufficient, for Rab9 binding. Mutation of these residues led to a significant decrease in Rab9 binding, but did not alter the global folding of the protein. The most impaired mutant was indistinguishable from wild-type TIP47 in its circular dichroism spectrum, and mutant proteins that showed decreased Rab9 binding retained full capacity to bind to MPR cytoplasmic domains. Closely related sequences in a related protein, adipophilin, did not confer Rab9 binding capacity to that protein. Partial proteolysis of TIP47 and TIP47 mutant proteins revealed subtle conformational differences, suggesting that residues 161-169 reside in a portion of TIP47 that is important for its conformation. These experiments reveal distinct binding domains for the Rab9 GTPase and MPR cytoplasmic domains in the cargo selection protein TIP47.

Figure 1

(A) Purified TIP47 mutant proteins. Twelve mutants were generated that contain three alanines in place of residues 152–187 as described in Methods. Proteins were expressed in E. coli and purified. Shown is a 12.5% SDS-polyacrylamide gel stained with Coomassie blue. Lane 1, TIP47 wild type. Lanes 2–12, TIP47 mutants 1–10 and 12. TIP47 mutant 11 is not shown. The specific three residues mutated in each mutant protein are listed in B. (B) Alanine scanning mutagenesis reveals specific residues in TIP47 important for Rab9 binding. His-tagged TIP47 mutants (200 nM) were assayed for Rab9 (200 nM) binding. The data were corrected for background binding obtained by using His-tagged GFP instead of His-TIP47 and are representative of at least two, and for most mutants three, independent experiments. The dashed line is included to aid comparison with the wild-type binding level observed. Shown below, anti-Rab9 immunoblot data used for this analysis.

Figure 2

(A) CD analysis of TIP47 (circles) and TIP47^SVV-AAA (triangles). Units for mean residue ellipticity [θ] are degrees·cm²⋅dmol⁻¹. (B) TIP47 and TIP47 mutants are fully active in binding MPR cytoplasmic domains. His-tagged TIP47 (200 nM) was incubated with GST–CI-MPRΔ12. Complexes were recovered on nickel beads and analyzed for the presence of GST–CI-MPR by immunoblot. Backgrounds were determined as in Fig. 1B and were subtracted. Shown are means and standard deviations of duplicate samples. Results are representative of three independent experiments. The dashed line is included as in Fig. 1.

Figure 3

(A) Sequence comparison of TIP47 and adipophilin. (B) Coomassie blue stained-SDS/PAGE analysis of purified adipophilin (*); adipophilin is the lower band in the doublet labeled, as determined by immunoblot. (C) Adipophilin does not bind Rab9. Binding was carried out as described in Methods; error bars represent the SE for duplicate measurements.

Figure 4

Proteolytic footprinting reveals minor conformational differences between TIP47 wild type and TIP47 SVV^167–169AAA. TIP47 wild-type and mutant proteins (3 μg) were incubated with varying amounts of protease for 30 min at 37°C. Reactions were stopped by addition of protease inhibitors and analyzed by 12.5% SDS/PAGE. Results are representative of two, and for wild-type TIP47 three, independent experiments. Shown below the gel are the microgram amounts of protease used.

Figure 5

TIP47 enhances Rab9 binding capacity, but is not a nucleotide exchange factor. Rab9 was incubated with 3 μM [³⁵S]GTPγS at 37°C for varying times with or without TIP47 (100 nM) as indicated. Rab9-nucleotide complexes were isolated on nitrocellulose filters and analyzed by scintillation counting. Shown are means and standard deviations of duplicate samples. Results are representative of four independent experiments.