Identification of residues in TIP47 essential for Rab9 binding - PubMed (original) (raw)
Identification of residues in TIP47 essential for Rab9 binding
John Hanna et al. Proc Natl Acad Sci U S A. 2002.
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.
Figures
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 TIP47SVV-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. 1_B_ 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 SVV167–169AAA. 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.
Similar articles
- Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells.
Barbero P, Bittova L, Pfeffer SR. Barbero P, et al. J Cell Biol. 2002 Feb 4;156(3):511-8. doi: 10.1083/jcb.200109030. Epub 2002 Feb 4. J Cell Biol. 2002. PMID: 11827983 Free PMC article. - Role of Rab9 GTPase in facilitating receptor recruitment by TIP47.
Carroll KS, Hanna J, Simon I, Krise J, Barbero P, Pfeffer SR. Carroll KS, et al. Science. 2001 May 18;292(5520):1373-6. doi: 10.1126/science.1056791. Science. 2001. PMID: 11359012 - Purification and analysis of TIP47 function in Rab9-dependent mannose 6-phosphate receptor trafficking.
Burguete AS, Sivars U, Pfeffer S. Burguete AS, et al. Methods Enzymol. 2005;403:357-66. doi: 10.1016/S0076-6879(05)03031-4. Methods Enzymol. 2005. PMID: 16473602 - [The double-play of PP17/TIP47].
Pauloin A, Ollivier-Bousquet M, Chanat E. Pauloin A, et al. Med Sci (Paris). 2004 Nov;20(11):1020-5. doi: 10.1051/medsci/200420111020. Med Sci (Paris). 2004. PMID: 15525499 Review. French. - [Function of PAT family proteins in the lipid metabolism].
Liu MF, Xu GH. Liu MF, et al. Sheng Li Ke Xue Jin Zhan. 2006 Apr;37(2):103-7. Sheng Li Ke Xue Jin Zhan. 2006. PMID: 16850611 Review. Chinese.
Cited by
- HIV-1 Hijacking of Host ATPases and GTPases That Control Protein Trafficking.
Tavares LA, Januário YC, daSilva LLP. Tavares LA, et al. Front Cell Dev Biol. 2021 Jul 8;9:622610. doi: 10.3389/fcell.2021.622610. eCollection 2021. Front Cell Dev Biol. 2021. PMID: 34307340 Free PMC article. Review. - Molecular modeling and molecular dynamics simulation study of the human Rab9 and RhoBTB3 C-terminus complex.
Junaid M, Muhseen ZT, Ullah A, Wadood A, Liu J, Zhang H. Junaid M, et al. Bioinformation. 2014 Dec 31;10(12):757-63. doi: 10.6026/97320630010757. eCollection 2014. Bioinformation. 2014. PMID: 25670879 Free PMC article. - Reevaluation of the requirement for TIP47 in human immunodeficiency virus type 1 envelope glycoprotein incorporation.
Checkley MA, Luttge BG, Mercredi PY, Kyere SK, Donlan J, Murakami T, Summers MF, Cocklin S, Freed EO. Checkley MA, et al. J Virol. 2013 Mar;87(6):3561-70. doi: 10.1128/JVI.03299-12. Epub 2013 Jan 16. J Virol. 2013. PMID: 23325685 Free PMC article. - Entry at the trans-face of the Golgi.
Pfeffer SR. Pfeffer SR. Cold Spring Harb Perspect Biol. 2011 Mar 1;3(3):a005272. doi: 10.1101/cshperspect.a005272. Cold Spring Harb Perspect Biol. 2011. PMID: 21421921 Free PMC article. Review. - Role of Rab GTPases in membrane traffic and cell physiology.
Hutagalung AH, Novick PJ. Hutagalung AH, et al. Physiol Rev. 2011 Jan;91(1):119-49. doi: 10.1152/physrev.00059.2009. Physiol Rev. 2011. PMID: 21248164 Free PMC article. Review.
References
- Kornfeld S, Mellman I. Annu Rev Cell Biol. 1989;5:483–525. - PubMed
- Kornfeld S. Annu Rev Biochem. 1992;61:307–330. - PubMed
- Diaz E, Pfeffer S R. Cell. 1998;93:433–443. - PubMed
- Carroll K S, Hanna J, Simon I, Krise J, Barbero P, Pfeffer S R. Science. 2001;292:1373–1376. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous