Abstract
Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [α-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.
References
Araki, S., Kaibuchi, K., Sasaki, T., Hata, Y., and Takai, Y. (1991). Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein. Mol. Cell. Biol.11, 1438–1447.Search in Google Scholar
Bordier, C. (1981). Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem.256, 1604–1607.10.1016/S0021-9258(19)69848-0Search in Google Scholar
Chen, D., Guo, J., Miki, T., Tachibana, M., and Gahl, W.A. (1997). Molecular cloning and characterization of rab27a and rab27b, novel human rab proteins shared by melanocytes and platelets. Biochem. Mol. Med.60, 27–37.10.1006/bmme.1996.2559Search in Google Scholar
Dobbs, L.G., Geppert, E.F., Williams, M.C., Greenleaf, R.D., and Mason, R.J. (1980). Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase. Biochim. Biophys. Acta618, 510–523.10.1016/0005-2760(80)90270-2Search in Google Scholar
Fischer von Mollard, G., Mignery, G.A., Baumert, M., Perin, M.S., Hanson, T.J., Burger, P.M., Jahn, R., and Sudhof, T.C. (1990). rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc. Natl. Acad. Sci. USA87, 1988–1992.10.1073/pnas.87.5.1988Search in Google Scholar PubMed PubMed Central
Fischer von Mollard, G., Sudhof, T.C., and Jahn, R. (1991). A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature349, 79–81.10.1038/349079a0Search in Google Scholar PubMed
Hunziker, W., and Peters, P.J. (1998). Rab17 localizes to recycling endosomes and regulates receptor-mediated transcytosis in epithelial cells. J. Biol. Chem.273, 15734–15741.10.1074/jbc.273.25.15734Search in Google Scholar PubMed
Jager, D., Stockert, E., Jager, E., Gure, A.O., Scanlan, M.J., Knuth, A., Old, L.J., and Chen, Y.T. (2000). Serological cloning of a melanocyte rab guanosine 5′-triphosphate-binding protein and a chromosome condensation protein from a melanoma complementary DNA library. Cancer Res.60, 3584–3591.Search in Google Scholar
Janknecht, R., de Martynoff, G., Lou, J., Hipskind, R.A., Nordheim, A., and Stunnenberg, H.G. (1991). Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc. Natl. Acad. Sci. USA88, 8972–8976.10.1073/pnas.88.20.8972Search in Google Scholar PubMed PubMed Central
Lapetina, E.G., and Reep, B.R. (1987). Specific binding of [α-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc. Natl. Acad. Sci. USA84, 2261–2265.10.1073/pnas.84.8.2261Search in Google Scholar PubMed PubMed Central
Loftus, S.K., Larson, D.M., Baxter, L.L., Antonellis, A., Chen, Y., Wu, X., Jiang, Y., Bittner, M., Hammer III, J.A., and Pavan, W.J. (2002). Mutation of melanosome protein RAB38 in chocolate mice. Proc. Natl. Acad. Sci. USA99, 4471–4476.10.1073/pnas.072087599Search in Google Scholar PubMed PubMed Central
Lowe, P.N., Sydenham, M., and Page, M.J. (1990). The Ha-ras protein, p21, is modified by a derivative of mevalonate and methyl-esterified when expressed in the insect/baculovirus system. Oncogene5, 1045–1048.Search in Google Scholar
Lutcke, A., Jansson, S., Parton, R.G., Chavrier, P., Valencia, A., Huber, L.A., Lehtonen, E., and Zerial, M. (1993). Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization. J. Cell Biol.121, 553–564.10.1083/jcb.121.3.553Search in Google Scholar PubMed PubMed Central
Mellman, I., and Warren, G. (2000). The road taken: past and future foundations of membrane traffic. Cell100, 99–112.10.1016/S0092-8674(00)81687-6Search in Google Scholar
Mizoguchi, A., Kim, S., Ueda, T., Kikuchi, A., Yorifuji, H., Hirokawa, N., and Takai, Y. (1990). Localization and intracellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain. J. Biol. Chem., 265, 11872–11879.10.1016/S0021-9258(19)38480-7Search in Google Scholar
Oiso, N., Riddle, S.R., Serikawa, T., Kuramoto, T., and Spritz, R.A. (2004). The rat Ruby (R) locus is Rab38: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain. Mamm. Genome15, 307–314.10.1007/s00335-004-2337-9Search in Google Scholar
Olkkonen, V.M., and Stenmark, H. (1997). Role of Rab GTPases in membrane traffic. Int. Rev. Cytol.176, 1–85.10.1016/S0074-7696(08)61608-3Search in Google Scholar
Osanai, K., Mason, R.J., and Voelker, D.R. (1998). Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol.19, 929–935.10.1165/ajrcmb.19.6.3292Search in Google Scholar
Osanai, K., Iguchi, M., Takahashi, K., Nambu, Y., Sakuma, T., Toga, H., Ohya, N., Shimizu, H., Fisher, J.H., and Voelker, D.R. (2001a). Expression and localization of a novel Rab small G protein (Rab38) in the rat lung. Am. J. Pathol.158, 1665–1675.10.1016/S0002-9440(10)64122-3Search in Google Scholar
Osanai, K., Mason, R.J., and Voelker, D.R. (2001b). Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells. Biochim. Biophys. Acta Mol. Cell Biol.1531, 222–229.10.1016/S1388-1981(01)00104-4Search in Google Scholar
Takai, Y., Sasaki, T., and Matozaki, T. (2001). Small GTP-binding proteins. Physiol. Rev.81, 153–208.10.1152/physrev.2001.81.1.153Search in Google Scholar PubMed
Toyofuku, K., Wada, I., Valencia, J.C., Kushimoto, T., Ferrans, V.J., and Hearing, V.J. (2001). Oculocutaneous albinism types 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wild-type proteins. FASEB J.15, 2149–2161.10.1096/fj.01-0216comSearch in Google Scholar PubMed
Wilson, S.M., Yip, R., Swing, D.A., O'Sullivan, T.N., Zhang, Y., Novak, E.K., Swank, R.T., Russell, L.B., Copeland, N.G., and Jenkins, N.A. (2000). A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc. Natl. Acad. Sci. USA97, 7933–7938.10.1073/pnas.140212797Search in Google Scholar PubMed PubMed Central
Zacchi, P., Stenmark, H., Parton, R.G., Orioli, D., Lim, F., Giner, A., Mellman, I., Zerial, M., and Murphy, C. (1998). Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells. J. Cell Biol.140, 1039–1053.10.1083/jcb.140.5.1039Search in Google Scholar PubMed PubMed Central
Zerial, M., and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol.2, 107–117.10.1038/35052055Search in Google Scholar PubMed
Zohar, M., Teramoto, H., Katz, B.Z., Yamada, K.M., and Gutkind, J.S. (1998). Effector domain mutants of Rho dissociate cytoskeletal changes from nuclear signaling and cellular transformation. Oncogene17, 991–998.10.1038/sj.onc.1202022Search in Google Scholar PubMed
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