Rab27b Association with Melanosomes: Dominant Negative Mutants Disrupt Melanosomal Movement
2002; Elsevier BV; Volume: 118; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2002.01754.x
ISSN1523-1747
AutoresYanru Chen, Preminda Samaraweera, Tung‐Tien Sun, Gert Kreibich, Seth J. Orlow,
Tópico(s)Retinal Development and Disorders
ResumoThe movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes. The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes. green fluorescent protein Griscelli syndrome lysosome-associated membrane glycoprotein 1 maltose binding protein trans-Golgi network tyrosinase-related protein 1 Rab proteins belong to a large family of small, membrane-associated GTPases; about 60 mammalian members in this family have already been identified (Pereira-Leal and Seabra, 2000Pereira-Leal J.B. Seabra M.C. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily.J Mol Biol. 2000; 301: 1077-1087https://doi.org/10.1006/jmbi.2000.4010Crossref PubMed Scopus (355) Google Scholar;Bock et al., 2001Bock J.B. Matern H.T. Peden A.A. Scheller R.H. A genomic perspective on membrane compartment organization.Nature. 2001; 409: 839-841Crossref PubMed Scopus (503) Google Scholar). Rab proteins may coordinate diverse cellular functions, including signal transduction, cytoskeletal organization, as well as the regulation of vesicular traffic and fusion in eukaryotic cells (Pfeffer, 1999Pfeffer S.R. Transport-vesicle targeting: tethers before snares.Nature Cell Biol. 1999; 1: E17-E22Crossref PubMed Scopus (355) Google Scholar;Zerial and McBride, 2001Zerial M. McBride H. Rab proteins as membrane organizers.Nature Rev Mol Cell Biology. 2001; 2: 107-117Crossref PubMed Scopus (2584) Google Scholar). These GTPases have been found to be associated with the cytoplasmic surface of distinct organelles, and with vesicles mediating transport between them. Specifically, Rab proteins are thought to be intimately involved in the process of docking and/or fusion of vesicles with their correct acceptor compartments. Regulation of specific targeting of vesicles by Rab proteins may occur at different stages of this transport process. First, Rab proteins may recruit factors involved in the biogenesis of the vesicles or they may mediate the tethering of motor molecules to the vesicle (Woodman, 1998Woodman P. Vesicle transport: more work for the Rabs?.Curr Biol. 1998; 8: R199-R201Abstract Full Text Full Text PDF PubMed Google Scholar). Rab proteins may also affect the targeting of vesicles to their appropriate destinations by interacting with Rab effector molecules that may mediate the interactions with cytoskeletal elements. For example, Rab6 mediates vesicle migration along the microtubular cytoskeleton, employing Rabkinesin-6 as the motor (Echard et al., 1998Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Interaction of a Golgi-associated kinesin-like protein with. Rab6.Science. 1998; 279: 580-585Crossref PubMed Scopus (401) Google Scholar). Rab3 has also been shown in vitro to interact with α-actinin through its effector Rabphilin-3A (Arribas et al., 1997Arribas M. Regazzi R. Garcia E. Wollheim C.B. De Camilli P. The stimulatory effect of rabphilin 3a on regulated exocytosis from insulin-secreting cells does not require an association-dissociation cycle with membranes mediated by Rab 3.European J Cell Biol. 1997; 74: 209-216PubMed Google Scholar). These results raise the intriguing possibility that Rab proteins regulate vesicle interactions with the cytoskeleton, thereby playing an active role in membrane trafficking (Pfeffer, 1999Pfeffer S.R. Transport-vesicle targeting: tethers before snares.Nature Cell Biol. 1999; 1: E17-E22Crossref PubMed Scopus (355) Google Scholar). Rab proteins may also tether vesicles to specific components at the fusion site via Rab-interacting molecules. Rabaptin-5, EEA1, and Rabenosyn-5 are effectors associated with Rab5. Through simultaneous or sequential interactions of these docking or membrane-tethering molecules with Rab5, they orchestrate vesicle fusion (Horiuchi et al., 1997Horiuchi H. Lippe R. McBride H.M. et al.A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar;Callaghan et al., 1999Callaghan J. Nixon S. Bucci C. Toh B.H. Stenmark H. Direct interaction of EEA1 with Rab5b.European J Biochem. 1999; 265: 361-366Crossref PubMed Scopus (48) Google Scholar;Christoforidis et al., 1999Christoforidis S. McBride H.M. Burgoyne R.D. Zerial M. The Rab5 effector EEA1 is a core component of endosome docking.Nature. 1999; 397: 621-625Crossref PubMed Scopus (633) Google Scholar;Nielsen et al., 2000Nielsen E. Christoforidis S. Uttenweiler-Joseph S. et al.Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain.J Cell Biol. 2000; 151: 601-612Crossref PubMed Scopus (264) Google Scholar). It was also shown that, once a vesicle has become tethered to its fusion site, Rab proteins may selectively activate the SNARE fusion machinery by removing the negative regulator Munc18 or n-Sec1 from the t-SNARE (Zhang et al., 2000Zhang W. Efanov A. Yang S.N. et al.Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic beta-cell.J Biol Chem. 2000; 275: 41521-41527Crossref PubMed Scopus (76) Google Scholar), allowing the free t-SNARE to form the triple-helical spindle required for vesicle fusion (Lin and Scheller, 2000Lin R.C. Scheller R.H. Mechanisms of synaptic vesicle exocytosis.Annu Rev Cell Dev Biology. 2000; 16: 19-49Crossref PubMed Scopus (406) Google Scholar). Rab proteins can be further subdivided into two families: one comprises the "housekeeping" Rab proteins, which are expressed in most cells and regulate basic vesicular transport common to all mammalian cells, like Rab5 regulating endosome transport (Woodman, 2000Woodman P. Biogenesis of the sorting endosome: the role of Rab5.Traffic. 2000; 9: 695-701Crossref Scopus (77) Google Scholar); the other includes specific Rabs, which are expressed in highly differentiated cells that carry out specialized functions. The restricted tissue distribution of certain Rab proteins may reflect specific requirements for the organization of membrane traffic in these cells. For example, the unique expression or enrichment of Rab37 in the MC-9 mast cell line and bone marrow cells suggests that Rab37 plays an important role in mast cell degranulation (Masuda et al., 2000Masuda E.S. Luo Y. Young C. et al.Rab37 is a novel mast cell specific GTPase localized to secretory granules.FEBS Lett. 2000; 470: 61-64Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It appears that Rab27b may also belong to the group of Rabs that are selectively expressed in highly specialized cells. Rab27b was originally isolated from human melanoma cells and melanocytes (Chen et al., 1997Chen D. Guo J. Miki T. Tachibana M. Gahl W.A. Molecular cloning and characterization of Rab27a and Rab27b, novel human Rab proteins shared by melanocytes and platelets.Biochem Mol Med. 1997; 60: 27-37Crossref PubMed Scopus (93) Google Scholar). Melanocytes are specialized cells in the skin and eye that express a unique elliptical membrane bounded organelle, the melanosome, which contains specific enzymes involved in melanin biosynthesis. Melanosomes contain an internal matrix upon which insoluble melanins are deposited. Based on morphologic studies, melanosomes have been described as undergoing four stages of development, namely, stage I and stage II premelanosomal compartments, partially melanized stage III melanosomes, and fully melanized stage IV melanosomes. Early models for melanosome biogenesis suggested that immature, endoplasmic-reticulum-derived organelles fuse with trans-Golgi network (TGN)-derived vesicles containing melanogenic components such as tyrosinase and TRP1/gp75 to form melanosomes (Quevedo et al., 1987Quevedo W.C. Fitzpatrick T.B. Szabó G. Jimbow K. Biology of melanocytes.in: Dermatology in General Medicine. McGraw-Hill, New York1987: 224-251Google Scholar;Orlow, 1998Orlow S.J. The biogenesis of melanosomes.in: James E. Nordlund J. The Pigmentary System: Physiology and Pathophysiology. Oxford University Press, New York1998: 97-116Google Scholar). Recent studies have suggested the alternative possibility that stage I premelanosomes are TGN-derived coated endosomes (Jimbow et al., 2000Jimbow K. Park J.S. Kato F. Hirosaki K. Toyofuku K. Hua C. Yamashita T. Assembly, target-signaling and intracellular transport of tyrosinase gene family proteins in the initial stage of melanosome biogenesis.Pigment Cell Res. 2000; 13: 222-229Crossref PubMed Scopus (102) Google Scholar;Raposo et al., 2001Raposo G. Tenzaa D. Murphyb D.M. Bersonb J.F. Marksb M.S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.J Cell Biol. 2001; 152: 809-824Crossref PubMed Scopus (332) Google Scholar). Pmel17 (gp100/silver locus protein) is the first well-characterized melanosomal protein that appears in early stage melanosomes (Kobayashi et al., 1994Kobayashi T. Urabe K. Orlow S.J. et al.The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function.J Biol Chem. 1994; 269: 29198-29205Abstract Full Text PDF PubMed Google Scholar). Other melanogenic enzymes such as tyrosinase and TRP1 are added subsequently. Melanosomes move bidirectionally along microtubules. At the cell periphery, myosin Va links melanosomes to the subcortical actin filaments in the dendritic extensions of melanocytes (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.J Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar) from which the melanosomes are taken up by recipient cells such as neighboring keratinocytes or are inserted into nascent hair shafts. Recent studies have shown that melanosome transport in melanocytes is highly regulated by Rab27a (Wilson et al., 2000Wilson S.M. Yip R. Swing D.A. et al.A mutation in Rab27a causes the vesicle transport defects observed in ashen mice.Proc Natl Acad Sci USA. 2000; 97: 7933-7938Crossref PubMed Scopus (322) Google Scholar;Hume et al., 2001Hume A. Collinson L. Rapak A. Gomes A. Hopkins C. Seabra M. Rab27a regulates the peripheral distribution of melanosomes in melanocytes.J Cell Biol. 2001; 152: F21-F24Crossref PubMed Scopus (265) Google Scholar;Wu et al., 2001Wu X. Rao K. Bowers M. Copeland N. Jenkins N. Hammer J.A. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle.J Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). Truncation of the RAB27A gene results in the phenotype of ashen mice and has been implicated in Griscelli syndrome (GS, MIM 214450). GS is a rare autosomal recessive disorder that results in pigmentary dilution of the skin and hair, the presence of large clumps of pigment in hair shafts, and an accumulation of melanosomes in the perinuclear region of melanocytes. Rab27a and myosin Va were both mapped to the GS locus at chromosome 15q21 but they are 1.6 cM apart from each other (Westbroek et al., 2001Westbroek W. Lambert J. Naeyaert J. The dilute locus and Griscelli syndrome: gateways towards a better understanding of melanosome transport.Pigment Cell Res. 2001; 14: 320-327https://doi.org/10.1034/j.1600-0749.2001.140503.xCrossref PubMed Scopus (32) Google Scholar). The phenotype of melanocytes in Rab27a-deficient ashen mice resembles that of melanocytes from myosin-Va-deficient dilute mice; they both fail to capture melanosomes in the cell periphery, resulting in the accumulation of mature melanosomes in the perinuclear region. Overexpression of the GTP-bound form but not the GDP form of Rab27a in ashen melanocytes rescued this defect. In addition, without functional Rab27a, myosin Va in ashen melanocytes does not colocalize with melanosomes (Wu et al., 2001Wu X. Rao K. Bowers M. Copeland N. Jenkins N. Hammer J.A. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle.J Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). It thus seems that Rab27a recruits myosin Va to the melanosome and that myosin Va then tethers the melanosomes to subcortical actin filaments at the periphery of the cell. A direct interaction between Rab27a and myosin Va was recently shown byHume et al., 2001Hume A. Collinson L. Rapak A. Gomes A. Hopkins C. Seabra M. Rab27a regulates the peripheral distribution of melanosomes in melanocytes.J Cell Biol. 2001; 152: F21-F24Crossref PubMed Scopus (265) Google Scholar) employing coimmunoprecipitation. Although mutations of either Rab27a or myosin Va produce the same phenotype of lightened coat color, they define two distinct subfamilies of GS. Patients with the RAB27A gene defects developed a hemophagocytic syndrome and Rab27a-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis required for immune homeostasis (Menasche et al., 2000Menasche G. Pastural E. Feldmann J. et al.Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome.Nature Genet. 2000; 25: 173-176Crossref PubMed Scopus (702) Google Scholar). The single Turkish patient with a myosin Va mutation, however, exhibited primary neurologic impairment without immune defects (Pastural et al., 1997Pastural E. Barrat F. Dufourcq-Lagelouse R. et al.Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosinVa gene.Nat Genet. 1997; 16: 289-292Crossref PubMed Scopus (345) Google Scholar), whereas Rab27a-deficient mice did not demonstrate any neurologic defect. These differences suggest that Rab27a may not be the sole partner of myosin Va. Sequence comparison with known Rabs indicates that Rab27a and Rab27b proteins comprise a melanocyte/platelet subfamily within the Rab family (Pereira-Leal and Seabra, 2000Pereira-Leal J.B. Seabra M.C. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily.J Mol Biol. 2000; 301: 1077-1087https://doi.org/10.1006/jmbi.2000.4010Crossref PubMed Scopus (355) Google Scholar). The expression of Rab27a and Rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized not only by the combination of pigment dilution and a platelet storage pool defect, but also with immunologic defects (Haddad et al., 2001Haddad E.K. Wu X. Hammer 3rd, J.A. Henkart P.A. Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice. [see comments]..J Cell Biol. 2001; 152: 835-842Crossref PubMed Scopus (202) Google Scholar). Despite the fact that Rab27b is the Rab protein most closely related to Rab27a, little new information emerged as it was cloned (Chen et al., 1997Chen D. Guo J. Miki T. Tachibana M. Gahl W.A. Molecular cloning and characterization of Rab27a and Rab27b, novel human Rab proteins shared by melanocytes and platelets.Biochem Mol Med. 1997; 60: 27-37Crossref PubMed Scopus (93) Google Scholar). The gene encoding human Rab27b was recently mapped to chromsome 18q21.1 (Ramalho et al., 2001Ramalho J. Tolmachova T. Hume A. McGuigan A. Gregory-Evans C. Huxley C. Seabra M. Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene.BMC Genet. 2001; 2: 2-13Crossref PubMed Scopus (39) Google Scholar), rather than chromosome 15 where Rab27a is located (Tolmachova et al., 1999Tolmachova T. Ramalho J.S. Anant J.S. Schultz R.A. Huxley C.M. Seabra M.C. Cloning, mapping and characterization of the human RAB27A gene.Gene. 1999; 239: 109-116https://doi.org/10.1016/s0378-1119(99)00371-6Crossref PubMed Scopus (0) Google Scholar). As these two highly homologous Rab proteins exhibit rather selective expression patterns, it is possible that they also possess functional overlap. On the other hand, Rab27b may also have a unique function because it cannot complement the ashen phenotype (Wilson et al., 2000Wilson S.M. Yip R. Swing D.A. et al.A mutation in Rab27a causes the vesicle transport defects observed in ashen mice.Proc Natl Acad Sci USA. 2000; 97: 7933-7938Crossref PubMed Scopus (322) Google Scholar). Here, we report on the tissue distribution of Rab27b in comparison with that of Rab27a by reverse transcription polymerase chain reaction (RT-PCR). The mRNA of Rab27b was present in melan-a cells and the protein was associated with melanosomes. Dominant negative forms of Rab27b do not associate with melanosomes. Overexpression of dominant-negative mutants of Rab27b not only caused redistribution of melanosomes from the cell periphery to the perinuclear region but also caused a diminution of dendrites in melan-a cells. Our results suggest that Rab27b may also play an important role in dendrite extension and in melanosome transport. Melan-a cells, cultured melanocytes from nonagouti black C57BL/6 J mice, were obtained from Dr. D. C. Bennett (St. George's Hospital, London, U.K.), and maintained as described previously (Bennett et al., 1987Bennett D.C. Cooper P.J. Hart I.R. A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumor promoter for growth.Int J Cancer. 1987; 39: 414-418Crossref PubMed Scopus (385) Google Scholar). The antiserum αPEP1 raised against tyrosinase-related protein 1 (Tyrp1) was from Dr. V. J. Hearing, National Cancer Institute, Bethesda, MD (Jimenez et al., 1991Jimenez M. Tsukamoto K. Hearing V.J. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes.J Biol Chem. 1991; 266: 1147-1156Abstract Full Text PDF PubMed Google Scholar). The antiserum Dil2 against mouse myosin Va (Wu et al., 1997Wu X. Bowers B. Wei Q. Kocher B. Hammer III, J.A. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor.J Cell Sci. 1997; 110: 847-859Crossref PubMed Google Scholar) was provided by Dr. John Hammer Jr. (National Heart, Lung and Blood Institute, Bethesda, MD). The sera were used in experiments without further purification. The rat monoclonal antibody 1D4B against murine lysosome-associated membrane glycoprotein 1 (Lamp1) (Hughes and August, 1981Hughes E.N. August J.T. Characterization of plasma membrane proteins identified by monoclonal antibodies.J Biol Chem. 1981; 256: 664-671Abstract Full Text PDF PubMed Google Scholar) was from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Cloning procedures were performed according to standard methods (DiMaio et al., 1982DiMaio D. Treisman R. Maniatis T. Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells.Proc Natl Acad Sci USA. 1982; 79: 4030-4034Crossref PubMed Scopus (90) Google Scholar). Enzymes and buffers were purchased from Roche (Nutley, NJ), Gibco (Gaithersburg, MD), or from New England Biolabs (Beverly, MA). The cDNA of bovine Rab27b was originally obtained by screening a bovine urothelium library, which was generated by Dr. Fang-Ming Deng in Dr. Tung-Tien Sun's laboratory (New York, NY). The coding region was cloned into pcDNA3.0 vector. Mutant forms of Rab27b, Rab27b-N133I, and Rab27b-Y29L were generated by PCR site-directed mutagenesis. Constructs were sequenced by the NYU DNA sequencing facility (Skirball Institute, New York, NY). In order to purify Rab27b for in vitro GTPase assays, Rab27b and its mutant forms were cloned into the pMALC2 vector (a gift from Dr. Mindong Ren, NYU, New York, NY) between the EcoR I and Sal I sites. The resulting plasmids encode fusion proteins that contain each Rab protein or its mutants attached to the C-terminus of the maltose binding protein, abbreviated MBP-Rab27b, MBP-N133I, and MBP-Y29L. Similarly, for the expression assay, Rab27b and its mutant forms were cloned into pEEGFPC2 vector (Stratagene, La Jolla, CA) between the EcoR I and Apa I sites. The resulting plasmids encode fusion proteins that contain each Rab protein or its mutants attached to the C-terminus of the enhanced green fluorescent protein, abbreviated EGFP-Rab27b, EGFP-N133I, and EGFP-Y29L. RNAs were isolated from 1-3 g of fresh tissue: urothelium scraped from bovine bladders or cultured cells from three plates (10 cm diameter) following the standard protocol (Gilman, 1997Gilman M. Preparation and analysis of RNA.in: Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1997: 414-452Google Scholar). The optical density at 260, 230, and 280 was measured to determine the amount and purity of the total RNA. Ratios of A260/A230 > 1.7 and A260/A280 > 1.7 were obtained. Intactness of the mRNA was inferred by intactness of 18S and 28S ribosomal RNA, as visualized on RNA agarose gels described in the next section. The first-strand cDNAs for the PCR reaction were synthesized from ≈25 ng of total RNA of each tissue per reaction using the AMV reverse transcriptase (Promega Cat. No. 3500). PCR was carried out using a PWO DNA polymerase (Roche, Nutley, NJ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to determine the amount of cDNA templates and the numbers of cycles within a linear range. The optimized cycle profile for Rab27a and Rab27b was one cycle, 94°C, 3 min; 25 cycles, 94°C, 1 min; 58°C, 45 s; 72°C, 1 min; one cycle, 72°C, 7 min. Primers for Rab27a and Rab27b PCR were 27a-forward, 5′-GCCACCATGTCCGAAACTGGATAAGCCAGC-3′; 27a-reverse, 5′-TCA AAG ATC TTA ATG TCT TCA ATG AGA C-3′; 27b-forward, ATGACCGATGGGGACTATGATTATCTG; 27b-reverse, 5′GGAGAAGTCAGCAGAGAAGAAATGTGC-3′. Mutated forms of bRab27b were constructed using a point mutagenesis PCR kit from Stratagene (La Jolla, CA). PCR cycles used were 12 cycles for a single base change and 16 cycles for two or three bases change for an amino acid change: 94°C, 1 min; 55°C, 1 min; 68°C, 2 min, for each 2 kb plasmid. Primers used to generate each mutant are as follows: N133I (no GTP/GDP binding, 397–399: AAC-ATC) forward, 5′-GTA TTA ATT GGC ATC AAG GCA GAC CTG CC-3′; reverse, 5′-GG CAG GTC TGC CTT GAT GCC AAT TAA TAC-3′5′-GG CAG GTC TGC CTT GAT GCC AAT TAA TAC-3′; Y29L (85–87: TAC-TTG) forward, 5′-AAG ACA ACA TTT CTT TAT CGA TTG ACA GAC-3′; reverse, 5′-GTC TGT CAA TCG ATA AAG AAA TGT TGT CTT CCC-3′. After the PCR, 1 µl of 5 U per µl of Dpn I restriction enzyme was added to each 50 µl PCR reaction and incubated at 37°C for 1 h to digest the circular plasmid template. 1 µl of the PCR-generated linearized plasmids was used to transform 50 µl of XL-1-blue cells (competency 1012 colonies per µg DNA). A single colony was inoculated on the second day for mini prep, followed by restriction enzyme digestion or sequencing to confirm the designed point mutation. Escherichia coli transformants containing MBP fusion plasmids were generated as described above. For purification, a 5 ml culture in LB containing ampicillin (50 µg per ml) was inoculated from the frozen stock and grown to saturation overnight at 37°C. The overnight cell culture was diluted into 1000 ml of medium in a 2 liter flask and grown under antibiotic selection at 37°C to an OD of 0.6 (2–4 h). IPTG was added (0.5 mM final) and the culture was grown at 37°C for an additional 3 h. After centrifuging the cell culture for 15 min, 3000 rpm at 4°C, the supernatant was decanted and the pellets were collected in a total of 10 ml of phosphate-buffered saline (PBS) containing 1 mM ethylenediamine tetraacetic acid (EDTA), 5 µg per ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, and were transferred to a 50 ml tube containing an equal volume of binding buffer pH 7.0 [10 mM NaPO4 buffer pH 7.2, 0.5 M NaCl, 1 mM sodium azide, 10 mM β-mercaptoethanol, 10 mM EDTA pH 8.0, and 10 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid]. After sonication, 5 ml of amylase resin was washed three times with the binding buffer and added to the supernatant mixing followed by rotation for 2 h at 4°C. To elute MBP fusion proteins, the amylase resin was collected by centrifugation (1–2 min, 2000 rpm) and washed three times with binding buffer. 3 ml of 10 mM maltose in the binding buffer was added to 2 ml of resin and mixed by inverting. Usually 1–2 mg per ml of purified protein were obtained (determined by Bradford assay), and stored at 4°C for up to 1 mo. For long-term storage at -70°C, the protein concentration was increased to 4 mg per ml by adding bovine serum albumin (BSA). Four micrograms of purified MBP-Rab27b or its mutants were incubated at 25°C for 10 min with 1.0 µCi of [α-32P]-GTP in 200 µl of a buffer containing 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), and 200 µg per ml BSA. MgCl2 was added to a final concentration of 10 mM and the solution was then diluted with 1 ml of 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 10 mM MgCl2 before passage through a nitrocellulose filter. The filter was then washed with 30 ml of the same dilution buffer and air dried, and the radioactivity retained in the filter was counted in a scintillation counter. This filter-binding assay allowed us to measure the GTP binding ability of proteins. The molar ratio of protein versus GTP reflects the native GTP binding capability of the GTPase because the protein retains its native conformation throughout the reaction. The determination of the GTPase activity is complicated by the fact that three different reactions affect the amount of [γ-32P]-GTP bound to the Rab protein. In addition to the real enzyme activity, which hydrolyzes GTP to GDP and releases free unlabeled phosphate, there are the intrinsic GTP falling-off rate and the GTP/GDP exchange rate that cannot be ignored in the reaction system, even in the absence of GTP exchange factors. The filter-binding assay can measure the amount of GTP initially loaded, but it cannot distinguish between GTP hydrolysis versus exchange or off-rate. Therefore, charcoal was used to absorb proteins and nucleotides, thus enabling us to monitor the released free γ-32P. Thirty micrograms of purified Rab27b or its mutants were incubated at 25°C for 10 min with 1.0 µCi [γ-32P]-GTP in 200 µl of a buffer containing 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 2 mM EDTA, 1 mM DTT, and 200 µg per ml BSA. MgCl2 was added to a final concentration of 10 mM. At different time intervals, equal aliquots of the reaction mixture (≈4 µg GTPase) were withdrawn and mixed with 0.5 ml of a cold 2% wt/vol charcoal suspension in 25 mM Tris-HCl, pH 7.5, 25 mM NaCl, 10 mM MgCl2. Fifty microliters of each supernatant was counted after sedimentation of the charcoal. Samples were normally boiled in the sample buffer containing 0.1 M DTT or 0.1 M β-mercaptoethanol before loading. For nonreducing gels, samples were boiled in the sample buffer without adding reducing reagent. Pre-stained protein molecular weight standards were obtained from Gibco/BRL (Gaithersburg, MD). Samples were then separated on 17% acrylamide gel and stained by 0.2% Coomassie
Referência(s)