Title: Rab6 Regulation of Rhodopsin Transport inDrosophila
Abstract: Rab6 is a GTP binding protein that regulates vesicular trafficking within the Golgi and post-Golgi compartments. We overexpressed wild-type, a GTPase defective (Q71L), and a guanine nucleotide binding defective (N125I) Rab6 protein inDrosophila photoreceptors to assess the in vivorole of Rab6 in the trafficking of rhodopsin and other proteins. Expression of Drab6Q71L greatly reduced the steady state levels of two rhodopsins, Rh1 and Rh3, whereasDrab6wt and Drab6N125I showed weaker effects. Analysis of a strain carrying Rh1 rhodopsin under a heat shock promoter showed thatDrab6Q71L, but notDrab6wt or Drab6N125I, prevents the maturation of rhodopsin beyond an immature 40 kDa form. Drab6Q71L is a GTPase defective mutant, indicating that anterograde transport of rhodopsin requires Rab6 GTPase function. The three Drab6 strains had no effect on the expression of several other photoreceptor proteins. TheDrab6Q71L photoreceptors show marked histological defects at young ages and degenerate over a two week time span. These results establish that rhodopsin is transported via a Rab6 regulated pathway and that defects in trafficking pathways lead to retinal degeneration. Rab6 is a GTP binding protein that regulates vesicular trafficking within the Golgi and post-Golgi compartments. We overexpressed wild-type, a GTPase defective (Q71L), and a guanine nucleotide binding defective (N125I) Rab6 protein inDrosophila photoreceptors to assess the in vivorole of Rab6 in the trafficking of rhodopsin and other proteins. Expression of Drab6Q71L greatly reduced the steady state levels of two rhodopsins, Rh1 and Rh3, whereasDrab6wt and Drab6N125I showed weaker effects. Analysis of a strain carrying Rh1 rhodopsin under a heat shock promoter showed thatDrab6Q71L, but notDrab6wt or Drab6N125I, prevents the maturation of rhodopsin beyond an immature 40 kDa form. Drab6Q71L is a GTPase defective mutant, indicating that anterograde transport of rhodopsin requires Rab6 GTPase function. The three Drab6 strains had no effect on the expression of several other photoreceptor proteins. TheDrab6Q71L photoreceptors show marked histological defects at young ages and degenerate over a two week time span. These results establish that rhodopsin is transported via a Rab6 regulated pathway and that defects in trafficking pathways lead to retinal degeneration. Members of the Rab family of small GTPases are localized in distinct subcellular compartments (1Novick P. Brennwald P. Cell. 1993; 75: 597-601Abstract Full Text PDF PubMed Scopus (316) Google Scholar), and within these compartments they regulate vesicular trafficking by cycling between GTP- and GDP-bound forms (2Zerial M. Stenmark H. Curr. Opin. Cell Biol. 1993; 5: 613-620Crossref PubMed Scopus (343) Google Scholar). A general model of Rab function has emerged in which a complex of Rab-GDP and guanine nucleotide dissociation inhibitor (GDI) 1The abbreviations used are: GDIguanine nucleotide dissociation inhibitorRT-PCRreverse transcription-polymerase chain reactionERGelectroretinographyPDAprolonged depolarizing afterpotentialERendoplasmic reticulum. is maintained in the cytosol. On binding of this complex to the donor membrane, GDI is displaced and GDP is exchanged for GTP. Rab-GTP is recruited onto the transport vesicle, which buds from the donor membrane and then associates with the target membrane. The Rab-GTP is thought to mediate fusion of the vesicle through interactions with effector molecules on the target membrane. It is not known whether GTP hydrolysis of the Rab-GTP is required for vesicle fusion or occurs after fusion. After GTP hydrolysis, Rab-GDP is retrieved from the target membrane by GDI and recycled to the donor membrane (3Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (666) Google Scholar, 4Pfeffer S. Curr. Opin. Cell Biol. 1994; 6: 522-526Crossref PubMed Scopus (296) Google Scholar, 5Nuoffer C. Balch W.E. Annu. Rev. Biochem. 1994; 63: 949-990Crossref PubMed Scopus (375) Google Scholar). guanine nucleotide dissociation inhibitor reverse transcription-polymerase chain reaction electroretinography prolonged depolarizing afterpotential endoplasmic reticulum. The study of point mutations in several rab genes affecting amino acids essential for guanine nucleotide interactions has documented the importance of the Rab-guanine nucleotide interactions in Rab function (6Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (423) Google Scholar, 7McConlogue L. Castellano F. deWit C. Schenk D. Maltese W.A. J. Biol Chem. 1996; 271: 1343-1348Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Martinez O. Antony C. Pehau-Arnaudet G. Berger E.G. Salamero J. Goud B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1828-1833Crossref PubMed Scopus (147) Google Scholar). The Rab6 protein is likely involved in intra-Golgi transport. A GTPase defective Rab6 greatly reduced transport of the proteins between cis/medial and late Golgi compartments in mammalian cell culture (9Martinez O. Schimdt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). More recently, Martinez et al. (8Martinez O. Antony C. Pehau-Arnaudet G. Berger E.G. Salamero J. Goud B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1828-1833Crossref PubMed Scopus (147) Google Scholar) found that overexpression of wild-type Rab6 and a GTPase-defective Rab6 redistributed a trans-Golgi protein to the ER membrane compartment. Biochemical studies using specialized cells, however, have suggested a role for Rab6 in post-Golgi transport. Rab6 is associated with post-Golgi vesicles in Torpedo marmorata electrocytes (10Jasmin B. Goud B. Camus G. Cartaud J. Neuroscience. 1992; 49: 849-855Crossref PubMed Scopus (27) Google Scholar), hypothalamic neuronal cells (11Tixier-Vidal A. Barret A. Picart R. Mayau V. Vogt D. Wiedenmann B. Goud B. J. Cell Sci. 1993; 105: 935-947PubMed Google Scholar), and frog retinal cells (12Deretic D. Papermaster D.S. J. Cell Sci. 1993; 106: 803-813PubMed Google Scholar). The studies with frog retinal cells suggested that the Rab6 protein is associated with rhodopsin-containing vesicles that exit from thetrans-Golgi on their way to the rod outer segment. The Drosophila photoreceptor provides an excellent experimental system to study Rab6 function in rhodopsin membrane trafficking, given the availability of mutations in rhodopsin and other genes that impede rhodopsin maturation. Many of these mutations result in age-dependent degeneration of photoreceptors, suggesting that correct rhodopsin trafficking is critical to maintenance of photoreceptor stability. Some human retinal diseases caused by rhodopsin mutations, may also be due to improper rhodopsin trafficking within the photoreceptor (13Sung C.-H. Makino C. Baylor D.A. Nathans J. J. Neurosci. 1994; 14: 5818-5833Crossref PubMed Google Scholar). In addition, an inherited form of choroideremia results from a defective Rab escort protein-1, establishing that defects in Rab protein function are involved in other human degenerative diseases (14Seabra M.C. Ho Y.K. Anant J.S. J. Biol. Chem. 1995; 270: 24420-24427Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). We established an in vivo system to study the role of Rab6 in the trafficking of rhodopsin and other photoreceptor proteins. Our results suggest that Rab6 is required for anterograde rhodopsin transport through the ER-Golgi complex. Further, defects in Rab6 trafficking also trigger retinal degeneration, strengthening the tie between defects in the rhodopsin maturation pathway and photoreceptor degeneration. Degenerate primers based on the conserved DTAGQ and NKXD sequence motifs found in all Rab proteins were used to RT-PCR amplify rab sequences from totalDrosophila RNA. RNA was isolated following methods of Cathala et al. (15Cathala G. Sacouret B. Mendez B. West M. Karin M. Martial J.A. Baxter J.D. DNA. 1983; 2: 329-335Crossref PubMed Scopus (1229) Google Scholar). RT-PCR reaction was performed as specified by the RT-PCR reaction kit manufacturer (Perkin-Elmer). The 170-base pair fragments recovered from these reactions were cloned and sequenced to identify the Drosophila rab6 sequence (Drab6). The 170-base pair fragment of Drab6 was then used to isolate the entire rab6 gene from aDrosophila genomic library. In situhybridizations, carried out as described by Ashburner (16Ashburner M. Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), placed the gene at 33C/D on the standard Drosophila salivary chromosome map. Site-directed mutagenesis was used to createDrab6N125I (AAC to ATC) andDrab6Q71L (CAG to CTG). The coding sequence of the two mutants and Drab6wt were placed under the control of the ninaE promoter and inserted in a P-element transformation vector (17Rubin G.M. Spradling A.C. Nucleic Acids Res. 1983; 11: 6341-6351Crossref PubMed Scopus (332) Google Scholar). Drosophila transgenic flies were made by standard means (18Spradling A.C. Roberts D.B. Drosophila: A Practical Approach. IRL Press Limited, Oxford, England1986: 175-196Google Scholar) using the null mutantninaE oI17 as the recipient strain. Four independent lines were obtained for Drab6wt andDrab6N125I and two independent lines were obtained for Drab6Q71L. All lines for each construct showed similar levels of Rab6 expression and rhodopsin defects as described in this paper. A polyclonal antibody toDrosophila Rab6 was generated using the GEX glutathioneS-transferase system (19Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). To generate the antibody, a 243-base pair region coding for a C-terminal region of Rab6 (amino acids 129–208) was placed in the pGEX-3 vector. The fusion protein was collected on glutathione-agarose beads and then recovered from the beads by eluting in 8 m urea, 1 mm glycine, 1 mm EDTA, 100 mm β-mercaptoethanol, 0.1m Tris, pH 8.0. The fusion protein was dialyzed overnight in 20 mm Tris, pH 8.0, and used to immunize mice. Proteins from fly heads were extracted in 60 mm Tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mm β-mercaptoethanol, and 0.1% bromphenol blue, separated by SDS-polyacrylamide gel electrophoresis (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) on 4–15%, 10%, or 12% gels, and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech) in 19 mm Tris, 150 mm glycine, 20% methanol. Proteins were detected using the polyclonal antibodies directed against Rh1 or Rh3 opsin, 2T. Washburn, M. Serikaku, and J. O'Tousa, unpublished data. RdgB (21Vihtelic T.S. Goebl M. Milligan S. O'Tousa J.E. Hyde D.R. J. Cell Biol. 1993; 122: 1013-1022Crossref PubMed Scopus (159) Google Scholar), Trp (22Montell C. Rubin G.M. Neuron. 1989; 2: 1313-1323Abstract Full Text PDF PubMed Scopus (872) Google Scholar), and NinaC (23Montell C. Rubin G.M. Cell. 1988; 52: 757-772Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Protein was detected using the ECL system (Amersham Pharmacia Biotech). To analyze the transient expression of rhodopsin, we used a stock designated hs-ninaE-hsv tag 14. This stock contained an HSV (epitope identified in herpes simplex virus glycoprotein D)-tagged rhodopsin under the heat shock promoter (24Kurada P. Tonini T.D. Serikaku M.A. Piccini J.P. O'Tousa J.E. Visual Neurosci. 1998; 15: 693-700Crossref PubMed Scopus (22) Google Scholar). Flies heterozygous for the tagged rhodopsin and the respective Drab6 P-transgene were heat shocked for 1 h at 37 °C and placed at room temperature (22 °C) for the indicated time. Protein separation, transfer, and detection were performed as stated above, using a monoclonal antibody directed against the HSV tag (Novagen Corp.). Electroretinography (ERG) recordings (as described in Larrivee et al. (25Larrivee D.C. Conrad S.K. Stephenson R.S. Pak W.L. J. Gen. Physiol. 1981; 78: 521-545Crossref PubMed Scopus (70) Google Scholar)) were performed on 2-day-old white eyed flies reared in a 12-h light/12-h dark cycle. Electron microscopy was performed as described by Washburn and O'Tousa (26Washburn T. O'Tousa J.E. Genetics. 1992; 130: 585-595Crossref PubMed Google Scholar). All genotypes were white eyed and maintained in a 12-h light/12-h dark cycle. The control, Drab6wt andDrab6N125I flies were homozygous forninaE +, whereas theDrab6Q71L flies were heterozygous forninaE +. 16 days oldDrab6wt flies heterozygous for rhodopsin were also sectioned and provided the same results (data not shown). We used a PCR-based approach to initiate a study of Drosophila rab6 and identified the rab6 gene previously named Drab6by Satoh et al. (27Satoh A. Tokunaga F. Ozaki K. FEBS Lett. 1997; 404: 65-69Crossref PubMed Scopus (24) Google Scholar). We created two Drab6mutations, the GTPase defective (Drab6Q71L) and the guanine nucleotide binding defective (Drab6N125I), by in vitromutagenesis. These two mutants and the wild-type (Drab6wt) coding sequences were placed under the control of the ninaE promoter to allow specific and high levels of expression only in the Drosophila R1−R6 class of photoreceptor cells (28Mismer D. Rubin G.M. Genetics. 1987; 116: 565-578Crossref PubMed Google Scholar). Protein blotting experiments usingDrosophila Rab6 antibody confirmed that transgenic flies carrying these genes made large amounts of the Rab6 proteins (Fig.1). The majority of the Rab6 protein in the transgenic flies possessed a higher apparent molecular mass than that seen in control wild-type flies, corresponding to a nonprenylated Rab6 protein (9Martinez O. Schimdt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). The lipid modified form of Rab6 was also easily observed in these transgenic flies. We estimate that 32 times more lipid-modified Rab6 protein was detected in flies expressing theDrab6wt constructs than in wild-type control flies. Similar high levels of modified Rab6 protein (37 times more protein in Drab6N125I, and 57 times more protein in Drab6Q71L) was observed in the other transgenic flies. To look for generalized defects in photoreceptor function because of expression of these Rab6 proteins, we assayed the light response by ERG (Fig. 2). All strains show a robust response to light stimuli. A prolonged depolarizing afterpotential (PDA) is seen in the ERG, on exposure to blue light, when a substantial amount of rhodopsin is converted to an active metarhodopsin form (29Pak W.L. Breakfield X.O. Neurogenetics: Genetic Approaches to the Nervous System. Elsevier-North Holland, New York1979: 67-99Google Scholar). Control flies generate a complete PDA, andDrab6wt and Drab6N125I flies show a slight defect in the PDA maintenance. Drab6Q71L flies completely lack a PDA. Given the importance of high rhodopsin levels in generating a PDA, these results suggested that Drab6wt andDrab6N125I have minor effects on rhodopsin expression, and Drab6Q71L flies possess much lower levels of rhodopsin. Rhodopsin protein levels were examined by Western blot analysis to assess the effects of the Drab6strains (Fig. 3). Rhodopsin levels are dramatically reduced in Drab6Q71L (12% of wild type). There is a more modest reduction in theDrab6wt and Drab6N125I flies (76 and 74% of wild-type levels, respectively).Figure 3Rhodopsin levels in wild-type,Drab6 wt , Drab6 N125I , and Drab6 Q71L flies. Shown is the immunoblot analysis of rhodopsin levels from head protein extracts of five flies tested. All flies were 2–3 days post-eclosion and heterozygous for a wild-type rhodopsin gene. Rhodopsin was detected by a polyclonal rhodopsin antibody. The estimation of protein levels are averages derived from densitometric analysis of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We analyzed the effects of the Drab6 strains in an experimental protocol designed to document defects in the rhodopsin maturation pathway (24Kurada P. Tonini T.D. Serikaku M.A. Piccini J.P. O'Tousa J.E. Visual Neurosci. 1998; 15: 693-700Crossref PubMed Scopus (22) Google Scholar, 30Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar) (see also "Experimental Procedures"). In these experiments, flies carried a rhodopsin gene tagged by an HSV epitope expressed from a heat shock promoter. Expression of this rhodopsin gene occurs only during a 37 °C heat shock, allowing the fate of rhodopsin synthesized during a restricted time window to be assessed. For the study here, we constructed strains carrying both the heat shock-controlled rhodopsin gene and each of the three Drab6 genes. A strain containing the heat shock-controlled rhodopsin gene but noDrab6 construct served as the control in these experiments. In the absence of heat shock, no HSV-tagged rhodopsin could be detected in protein blotting experiments (Fig.4 A). Two h following the heat shock, the rhodopsin is detected as a 40-kDa species (open arrow) as well as several slightly higher molecular mass forms. 14.5 h after the pulse, rhodopsin is still present in the 40-kDa form but now is also detected in lower molecular mass bands (35–38 kDa). At 24 h after the heat shock, most of the rhodopsin is found in the 35-kDa form (filled arrow). This 35-kDa form has the same mobility as the major species of rhodopsin found in flies expressing the HSV-tagged rhodopsin from the ninaE promoter, hence we consider it the mature form. The strains containing theDrab6wt and Drab6N125I genes had the same profile as the control strain (Fig. 4, Cand D). The Drab6Q71L flies, however, showed defects in rhodopsin maturation (Fig. 4 B). Two h after heat shock, the majority of the rhodopsin was detected in the 40-kDa form, as expected from the analysis of the other strains. However, at 14.5, 24, and 48 h after heat shock, the 40-kDa rhodopsin remained as the predominant species. The data establish that the Drab6Q71L mutant is defective in processing the immature 40-kDa rhodopsin species into the mature 35-kDa form. In Drosophila six different opsins are expressed in subsets of the photoreceptor cells. To test the effects of the Drab6 constructs on a different rhodopsin, we misexpressed the Rh3 rhodopsin in R1−R6 photoreceptor cells (31Feiler R. Bjornson R. Kirshfeld K. Mismer D. Rubin G.M. Smith D.P. Socolich M. Zuker C.S. J. Neurosci. 1992; 12: 3862-3868Crossref PubMed Google Scholar). Western blot analysis of these strains (Fig.5 A) showed that Rh3 protein levels were reduced in all three Drab6 transgenic strains compared with controls. As with expression of the Rh1 (NinaE) protein, Rh3 levels were most reduced in the Drab6Q71L flies, with the other two lines showing a significant, but smaller, reduction of protein. We examined the protein levels of two other photoreceptor membrane proteins involved in phototransduction to determine whether theDrab6Q71L effect was specific to rhodopsin. RdgB is a membrane protein that is localized to the photoreceptor sub rhabdomeric-cisternae (21Vihtelic T.S. Goebl M. Milligan S. O'Tousa J.E. Hyde D.R. J. Cell Biol. 1993; 122: 1013-1022Crossref PubMed Scopus (159) Google Scholar), and Trp is a Ca2+ channel protein that co-localizes with rhodopsin in the rhabdomeres (32Niemeyer B.A. Suzuki E. Scott K. Jalink K. Zuker C.S. Cell. 1996; 85: 651-659Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Neither RdgB (Fig. 5 B) nor Trp (Fig. 5 C) protein levels were dramatically affected in any Drab6 strain. Similarly, the protein levels of the membrane-associatedninaC-encoded cytoskeletal photoreceptor proteins (Fig.5 D) were not affected. Electron microscopy was carried out to assess the changes in photoreceptor ultrastructure caused by overexpression of theDrab6 genes. Photoreceptors R1−R6 express theninaE-encoded Rh1 rhodopsin and, therefore, also express theDrab6 genes constructed in this study. The R7 cell, shown in Fig. 6, A-C, will not express the Drab6 transgenes and therefore serves as a convenient control in all micrographs. Three-day old control photoreceptors are shown in Fig. 6 A. Drab6wt andDrab6N125I R1−R6 photoreceptors (data not shown) are similar in structure to the control. Drab6Q71L flies (Fig. 6 B), however, show a marked reduction in the R1−R6 rhabdomeres volume. The area of the R1−R6 rhabdomeres in the Drab6Q71L rhabdomeres is similar in size to the R7 rhabdomere, even though the wild-type R1−R6 rhabdomeres are 70% larger (33Leonard D.S. Bowman V.D. Ready D.F. Pak W.L. J. Neurobiol. 1992; 23: 605-626Crossref PubMed Scopus (85) Google Scholar). Drab6Q71L R1−R6 cells possess an abnormal accumulation of membranes at the base of the rhabdomeres (arrow in Fig. 6 D). Some R1−R6 photoreceptors show loosely organized rhabdomeric membranes (arrow in Fig.6 E). Another striking feature is the frequent appearance of "whorl" membranes (34Blest A.D. Williams T.P. Baker B.W. The Effects of Constant Light on Visual Processes. Plenum Press, New York, NY1980: 217-245Crossref Google Scholar) within the cell (arrow in Fig.6 F). Histological analysis on older flies indicated that overexpression of all three Drab6 genes triggered retinal degeneration. A cross section of an ommatidial unit of a 16-day oldDrab6Q71L is shown in Fig. 6 C (data not shown for Drab6wt andDrab6N125I). Some R1−R6 photoreceptors of all the strains lacked rhabdomeric membranes (cell bodies marked witharrowheads in Fig. 6 C). A major objective of this study was to investigate the role of Rab6 in rhodopsin maturation. Protein blotting experiments and ERG analysis established thatDrab6Q71L flies possessed about 12% of the wild-type steady state levels of rhodopsin. Drab6wt and Drab6N125I flies possess about 75% of the wild-type rhodopsin levels. The only deficit in the ERG traces can be attributed to the reduction in rhodopsin content, indicating that overexpression of the Rab6 proteins did not have a debilitating effect on the physiology of these photoreceptor cells. Analysis of rhodopsin transport using a heat shock-regulated promoter demonstrated that in wild-type, Drab6wt, andDrab6N125I photoreceptors, rhodopsin matures to its final 35-kDa form within 24 h. In contrast, rhodopsin maturation is severely impaired in Drab6Q71L, showing little progression beyond the 40-kDa intermediate form. Previous work established that the 40-kDa rhodopsin is a high mannose intermediate found within the ER. The 40-kDa rhodopsin requires theninaA encoded cyclophilin (35Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar) and retinal addition (36Ozaki K. Nagatani H. Ozaki M. Tokunaga F. Neuron. 1993; 10: 1113-1119Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 37Huber A. Wolfrum U. Paulsen R. Eur. J. Cell Biol. 1994; 63: 219-229PubMed Google Scholar) to exit the ER. Our results show that Rab6Q71L blocks rhodopsin transport prior to its progression into the cis or medial Golgi compartment that contains the mannosidase which acts on the high mannose rhodopsin intermediate (38Pelham H.R.B. Annu. Rev. Cell Biol. 1989; 5: 1-23Crossref PubMed Scopus (542) Google Scholar, 39Rothman J.E. Orci L. FASEB J. 1990; 4: 1460-1468Crossref PubMed Scopus (109) Google Scholar). These results are consistent with a role of Rab6 in intra-Golgi transport. Although we have no data suggesting Rab6 in post-Golgi events as suggested by a study on frog retinal cells (12Deretic D. Papermaster D.S. J. Cell Sci. 1993; 106: 803-813PubMed Google Scholar), our analysis does not rule out a second independent role of Rab6. A recent study showed that transient expression of Rab1N124I protein prevents rhodopsin maturation beyond the 40-kDa intermediate (40Satoh A.K. Tokunaga F. Kawamura S. Ozaki K. J. Cell Sci. 1997; 110: 2943-2953PubMed Google Scholar), similar to the phenotype observed in theDrab6Q71L mutant. It is striking that dominant mutants of the first two Rab proteins studied in theDrosophila photoreceptor appear to affect similar stages of rhodopsin maturation. However, rhodopsin likely remains in a 40-kDa form as it trafficks from the ER to the cis or medial Golgi where modifications of the oligosaccharide side chain is thought to occur. Therefore, multiple Rabs, including the Rab1 and Rab6 proteins, may be required in these steps. The expression of Rh3 rhodopsin was also markedly reduced in the Drab6Q71L flies, and smaller effects were seen in the two other Drab6 strains. On the other hand, none of the Drab6 strains affected the levels of other photoreceptor proteins tested. These results suggest that rhodopsin transport is more sensitive to defects in the Rab6-regulated pathway, with alternative maturation pathways available for other photoreceptor membrane proteins. An alternative explanation, that RdgB and Trp are transported via the Rab6 pathway but nonetheless are maintained at normal levels, is only plausible if the stability of these proteins is dramatically increased inDrab6Q71L mutant photoreceptors. Resolution of these issues will likely require the identification and analysis of anin vivo loss of function rab6 mutant. By analogy with point mutations of rab6 (9Martinez O. Schimdt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar) and other rab genes (6Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (423) Google Scholar,41Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (778) Google Scholar), the Gln to Leu change prevents GTP hydrolysis. Therefore the Rab6Q71L mutant protein will always be bound to GTP. Drab6Q71L is a potent inhibitor of rhodopsin protein transport, which is consistent with the behavior of this mutation in other Rab6 studies (8Martinez O. Antony C. Pehau-Arnaudet G. Berger E.G. Salamero J. Goud B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1828-1833Crossref PubMed Scopus (147) Google Scholar, 9Martinez O. Schimdt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). If GTP hydrolysis is required for vesicle fusion, as proposed for Rab3a (42Johannes L. Lledo P.-M. Roa M. Vincent J.-D. Henry J.-P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (187) Google Scholar),Drab6Q71L is expected to prevent the fusion of vesicles with their target membrane. Our results showing that theDrab6Q71L form inhibits rhodopsin transport is consistent with a role for GTP hydrolysis to promote anterograde transport of rhodopsin-bearing vesicles. Alternatively, Rab6 in its GTP form could be a positive regulator of the retrograde transport, as proposed by Martinez et al. (8Martinez O. Antony C. Pehau-Arnaudet G. Berger E.G. Salamero J. Goud B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1828-1833Crossref PubMed Scopus (147) Google Scholar, 9Martinez O. Schimdt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). According to this notion, Drab6Q71L could increase the flow of retrograde transport and indirectly disrupt the anterograde pathway, resulting in inhibition of protein transport. However, this model was originally proposed to rationalize results showing that Rab6wt has similar effects as Rab6Q72L that were not confirmed in our experiments. We also documented an inhibition of Rh1 and Rh3 rhodopsin expression in the Drab6wt andDrab6N125I strains. However, the heat shock analysis indicates that Drab6wt andDrab6N125I have little or no inhibitory effects on the maturation of the 40- to the 35-kDa form of Rh1 rhodopsin. Thus, the mechanism of Drab6wt andDrab6N125I action is distinct from that ofDrab6Q71L. The Drab6wt and Drab6N125I proteins might have an effect on later stages of rhodopsin maturation, but it is also possible that the reduction in rhodopsin is a consequence of secondary effects associated with the overexpression of these proteins. All Rab proteins require isoprenylation to be functional (43Magee T. Newman C. Trends Cell Biol. 1992; 2: 318-323Abstract Full Text PDF PubMed Scopus (62) Google Scholar). When we overexpress Rab6 in photoreceptors, 25–35% of the protein is isoprenylated. The failure to completely modify the large amount of Rab6 found in these flies suggests that overexpression has overwhelmed the Rab geranylgeranyl transferase pathway responsible for the prenylation of all Rab proteins (46Porter J.A. Hicks J.L. Williams D.S. Montell C. J. Cell Biol. 1992; 116: 683-693Crossref PubMed Scopus (126) Google Scholar). Therefore, overexpression of Rab6 may also inhibit the modification, and therefore the activity, of other Rab proteins. Thus, the defects seen in photoreceptors overexpressing Rab6wt or Rab6N125I may not be directly attributable to the altered Rab6 activity. It is surprising that Drab6wt andDrab6N125I have similar effects. The Asn to Ile mutation is thought to create a defect in guanine nucleotide binding. In mammalian cell culture, the Asn to Ile mutant of Rab2 and Rab3a proteins show similar inhibitory effect on secretion as observed for the Gln to Leu mutations (6Tisdale E.J. Bourne J.R. Khosravi-Far R. Der C.J. Balch W.E. J. Cell Biol. 1992; 119: 749-761Crossref PubMed Scopus (423) Google Scholar, 42Johannes L. Lledo P.-M. Roa M. Vincent J.-D. Henry J.-P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (187) Google Scholar). On the other hand, the Asn to Ile mutation in rab6 increased secretion rate (7McConlogue L. Castellano F. deWit C. Schenk D. Maltese W.A. J. Biol Chem. 1996; 271: 1343-1348Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The lack of a mutant phenotype in our studies does not result from Rab6N125I protein instability since protein immunoblots show high levels of this protein. It appears that the Rab6N125I protein, perhaps because of lack of nucleotide binding, is unable to participate in the rab6 cycle. Overexpression of any form of Rab6 caused retinal degeneration, but the rate and severity of degeneration depended upon the form of Rab6. At young ages,Drab6Q71L photoreceptors already show structural differences that distinguish it from Drab6wt andDrab6N125I photoreceptors. The most striking difference is a much smaller volume occupied by the R1−R6 rhabdomeres. This phenotype is shared with mutant ninaE (33Leonard D.S. Bowman V.D. Ready D.F. Pak W.L. J. Neurobiol. 1992; 23: 605-626Crossref PubMed Scopus (85) Google Scholar, 44O'Tousa J.E. Leonard D.S. Pak W.L. J. Neurogenet. 1989; 6: 41-52Crossref PubMed Scopus (82) Google Scholar),ninaA (35Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar), ninaC (45Matsumoto H. Isono K. Pye Q. Pak W.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 985-989Crossref PubMed Scopus (56) Google Scholar, 46Porter J.A. Hicks J.L. Williams D.S. Montell C. J. Cell Biol. 1992; 116: 683-693Crossref PubMed Scopus (126) Google Scholar), as well as vitamin A deprived flies (47Sapp R.J. Christianson J.S. Maier L. Studer K. Stark W.S. Exp. Eye Res. 1991; 53: 73-79Crossref PubMed Scopus (21) Google Scholar). All these flies possess reduced rhodopsin content, suggesting that the reduced size of the rhabdomere in theDrab6Q71L mutant is likely the result of poor rhodopsin maturation. The Drab6Q71L photoreceptors exhibit other ultrastructural defects, most notably an accumulation of disorganized membranes within the cytoplasm as well as "whorl" membranes thought to represent membrane recycling processes (34Blest A.D. Williams T.P. Baker B.W. The Effects of Constant Light on Visual Processes. Plenum Press, New York, NY1980: 217-245Crossref Google Scholar). Satoh et al. (40Satoh A.K. Tokunaga F. Kawamura S. Ozaki K. J. Cell Sci. 1997; 110: 2943-2953PubMed Google Scholar) documented a similar phenotype in the Drosophila rab1N124I mutant. Consistent results are also obtained in mammalian cell culture. Martinez et al. (8Martinez O. Antony C. Pehau-Arnaudet G. Berger E.G. Salamero J. Goud B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1828-1833Crossref PubMed Scopus (147) Google Scholar) documented that overexpression of the rab6Q72L mutant allows the mixing of ER and Golgi membrane compartments, and morphological changes of the ER/Golgi are noted in other studies using lovastatin to limit prenylation of Rab proteins (48Ivessa N.E. Gravotta D. De Lemos-Chiarandini C. Kreibich G. J. Biol. Chem. 1997; 272: 20828-20834Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Thus, the abnormal membrane accumulation documented inDrab6Q71L photoreceptors may result from abnormal Golgi organization, and the defects in rhodopsin maturation may be a secondary consequence of this defect. On the other hand, our data are not compatible with a catastrophic defect in ER-Golgi transport in Drab6Q71L photoreceptors, as these photoreceptors retain normal physiological function, and other membrane proteins are detected at normal levels. Dominant rhodopsin mutants cause age-dependent retinal degeneration as a result of defects in rhodopsin transport (30Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar, 49Kurada P. O'Tousa J.E. Neuron. 1995; 14: 571-579Abstract Full Text PDF PubMed Scopus (115) Google Scholar). We initiated this study to examine the role of Rab6 in rhodopsin transport and to explore an in vivo experimental system to study the trafficking of rhodopsin. Our results establish the importance of Rab6-regulated trafficking mechanisms in both rhodopsin biogenesis and maintenance of photoreceptor morphology and function. We thank Sheila Adams for assistance with histology, Kathleen Mitchell and Tim Tonini for help in construction of the transgenic Drosophila strains, Paul Vieta for help with ERGs, Michael Nonet and Koichi Ozaki for sharing their Rab6 sequence data prior to publication, Craig Montell and David Hyde for antibodies, Steve Britt for the Rh3 transgenic strain, and Tracy Washburn and Michael Zimmerman for critical reading of this manuscript.