PRA Isoforms Are Targeted to Distinct Membrane Compartments

The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms. The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms. endoplasmic reticulum prenylated Rab acceptor hemagglutinin polymerase chain reaction guanyl-5′-yl thiophosphate guanosine 5′-3-O-(thio)triphosphate guanyl-5′-yl thiophosphate Chinese hamster ovary Rab GTPases are a family of 20–29-kDa Ras-like GTPases localized to unique intracellular organelles (1Chavrier P. Vingron M. Sander C. Simons K. Zerial M. Mol. Cell. Biol. 1990; 10: 6578-6585Crossref PubMed Scopus (176) Google Scholar, 2Elferink L.A. Anzai K. Scheller R.H. J. Biol. Chem. 1992; 267: 5768-5775Abstract Full Text PDF PubMed Google Scholar, 3Ngsee J.K. Elferink L.A. Scheller R.H. J. Biol. Chem. 1991; 266: 2675-2680Abstract Full Text PDF PubMed Google Scholar, 4Matteoli M. Takei K. Cameron R. Hurlbut P. Johnston P.A. Sudhof T.C. Jahn R. De Camilli P. J. 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Guanine nucleotide exchange occurs at the membrane and is catalyzed by a guanine nucleotide exchange factor of which a Rab3-specific (14Wada M. Nakanishi H. Satoh A. Hirano H. Obaishi H. Matsuura Y. Takai Y. J. Biol. Chem. 1997; 272: 3875-3878Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and the yeast Sec4p-specific form have been identified (15Walch-Solimena C. Collins R.N. Novick P.J. J. Cell Biol. 1997; 137: 1495-1509Crossref PubMed Scopus (270) Google Scholar, 16Elkind N.B. Walch-Solimena C. Novick P.J. J. Cell Biol. 2000; 149: 95-110Crossref PubMed Scopus (44) Google Scholar). The low intrinsic GTPase activity requires catalysis by a GTPase activating protein of which a Rab3A-specific (17Fukui K. Sasaki T. Imazumi K. Matsuura Y. Nakanishi H. Takai Y. J. Biol. Chem. 1997; 272: 4655-4658Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and yeast Ypt-specific (18Albert S. Gallwitz D. J. Biol. Chem. 1999; 274: 33186-33189Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Strom M. Vollmer P. Tan T.J. Gallwitz D. Nature. 1993; 361: 736-739Crossref PubMed Scopus (146) Google Scholar) forms have been identified. In ER1 to Golgi membrane trafficking, donor vesicles are tethered to the acceptor membrane compartment prior to SNARE-mediated fusion (20Cao X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (291) Google Scholar, 21Cao X. Barlowe C. J. Cell Biol. 2000; 149: 55-66Crossref PubMed Scopus (117) Google Scholar, 22Brigance W.T. Barlowe C. Graham T.R. Mol. Biol. Cell. 2000; 11: 171-182Crossref PubMed Scopus (46) Google Scholar, 23VanRheenen S.M. Cao X. Sapperstein S.K. Chiang E.C. Lupashin V.V. Barlowe C. Waters M.G. J. Cell Biol. 1999; 147: 729-742Crossref PubMed Scopus (107) Google Scholar). Rab has been shown to mediate the recruitment of a cytosolic tethering protein, Uso1p, to the membranes. Another large protein complex called TRAPP may also be involved in this transport step (24Sacher M. Jiang Y. Barrowman J. Scarpa A. Burston J. Zhang L. Schieltz D. Yates 3rd, J.R. Abeliovich H. Ferro-Novick S. EMBO J. 1998; 17: 2494-2503Crossref PubMed Scopus (238) Google Scholar). Tethering factors have been identified in other transport steps, suggesting conservation of this basic underlying process (25Christoforidis S. McBride H.M. Burgoyne R.D. Zerial M. Nature. 1999; 397: 621-625Crossref PubMed Scopus (661) Google Scholar, 26Barr F.A. Nakamura N. Warren G. EMBO J. 1998; 17: 3258-3268Crossref PubMed Scopus (205) Google Scholar, 27Guo W. Roth D. Walch-Solimena C. Novick P. EMBO J. 1999; 18: 1071-1080Crossref PubMed Scopus (499) Google Scholar). Once docked, however, vesicle fusion is no longer dependent on Rab, but requires the SNARE proteins. Thus, stability of docked vesicles may represent a number of distinct molecular states: from molecular interactions that merely hold the two membranes in close proximity to those needed to trigger bilayer fusion. Studies on synaptic vesicles have shown that docked vesicles will undock about once every 2 min, a rate that is faster than spontaneous fusion (28Murthy V.N. Stevens C.F. Nat. Neurosci. 1999; 2: 503-507Crossref PubMed Scopus (189) Google Scholar). Thus, reversibility of vesicle tethering and docking provides a time constraint on subsequent steps. Little is known on the mechanism of Rab localization to a specific membrane compartment. We have recently isolated a Rab-interacting protein called prenylated Rabacceptor or PRA1 (29Martincic I. Peralta M.E. Ngsee J.K. J. Biol. Chem. 1997; 272: 26991-26998Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). PRA1 interacts with both Rab and VAMP2 but not as a stable trimeric complex. In fact, binding of PRA1 to VAMP2 can be displaced by Rab3A, suggesting that PRA1 participates in the sequential assembly of protein by associating and dissociating from the Rab and VAMP2. Moreover, PRA1 inhibits the removal of Rab from the membrane by GDI (30Hutt D.M. Da-Silva L.F. Chang L.H. Prosser D.C. Ngsee J.K. J. Biol. Chem. 2000; 275: 18511-18519Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Thus, the recycling of Rab depends on the opposing action of PRA1 and GDI, with PRA1 favoring membrane retention and GDI favoring solubilization in the cytosol. The existence of multiple isoforms seems to be a common theme in proteins involved in the various vesicle transport steps. Multiple isoforms of VAMP and syntaxin supports the concept that distinct members mediate specific membrane trafficking steps (31Advani R.J. Bae H.R. Bock J.B. Chao D.S. Doung Y.C. Prekeris R. Yoo J.S. Scheller R.H. J. Biol. Chem. 1998; 273: 10317-10324Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 32Advani R.J. Yang B. Prekeris R. Lee K.C. Klumperman J. Scheller R.H. J. Cell Biol. 1999; 146: 765-776Crossref PubMed Scopus (155) Google Scholar, 33Zeng Q. Subramaniam V.N. Wong S.H. Tang B.L. Parton R.G. Rea S. James D.E. Hong W. Mol. Biol. Cell. 1998; 9: 2423-2437Crossref PubMed Scopus (60) Google Scholar). We describe here the isolation of a second PRA isoform that shares many similarities to PRA1 including overall physical properties, tissue distribution, and broad binding specificity toward the Rab GTPases. However, it differs from PRA1 in its subcellular localization. Whereas PRA1 was localized predominantly in the Golgi complex, PRA2 was found in the ER compartment. Moreover, we showed that the localization signal resides in the COOH-terminal region of the proteins. A EST data base search using the conserved domain of the rat PRA1 resulted in a number of similar clones, most notably that with accession numbers AW519501 and AA051031. The rat PRA2 was subcloned into a vector containing the hemagglutinin (HA) tag by ligation between the StuI and XbaI sites. PRA2 was PCR-amplified from a rat brain cDNA library using the following oligonucleotides: 5′-TAAGGCCTATGGACGTGAACCTCG-3′ and 5′-GCTCTAGATTACTCCCTCGCTTTGCTGA-3′. The resulting PCR fragment was sequenced. A 32P-labeled full-length PRA2 probe was prepared using random hexamer labeling (Life Technologies). A multiple tissue blot (CLONTECH) containing poly(A)+-selected mRNA of various rat tissues was hybridized with 1 × 106 cpm/ml of32P-labeled PRA2 in 10 ml of hybridization solution (5 × SSPE, 10 × Denhardt's solution, 100 μg/ml sheared salmon sperm DNA, 50%formamide, and 0.4%SDS) at 42 °C overnight. PRA2 was subcloned into the yeast two-hybrid prey vector, pGAD424X using PRA2.E, 5′-CGAGAATTCTTACTCCCTGGCTTTGCTGAT-3′ and PRA2.X2, 5′-GCCTCGAGTTACTCCCTSGCTTTGCTGA-3′. The VAMP1, VAMP2, and VAMP3 bait plasmids were described previously (29Martincic I. Peralta M.E. Ngsee J.K. J. Biol. Chem. 1997; 272: 26991-26998Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The HA-tagged PRA2 was subcloned into pQE10 (Qiagen) between theBamHI and the HindIII sites after PCR amplification using the following oligonucleotides: 5′-GCGGATCCTATGTACCCATACGATG-3′ and 5′-GACAAGCTTACTCCCTGGCTTTGC-3′. The 6xHis-tagged PRA2 was purified the same way as the PRA1 using Ni-NTA resin as described (29Martincic I. Peralta M.E. Ngsee J.K. J. Biol. Chem. 1997; 272: 26991-26998Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The Rab GTPases were also subcloned into the 6xHis-tagged pQE41 (Qiagen) between the BamHI and SphI sites. Rab1A was PCR-amplified using the following oligonucleotides: 5′-TGTGGATCCATGTCCAGCATGAATCCCGAA-3′ and 5′-CTAGGCATGCTTAGCAGCAGCCTCCACCTG-3′. The 6xHis-tagged Rab1A fragment was ligated into pYES2 (Invitrogen) between the EcoRI andSphI sites. The plasmid was then transformed into the yeast INVSc1 strain with Li acetate (34Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1704) Google Scholar). To purify the 6xHis-tagged Rab1A, the transformed yeast was grown to saturation in 500 ml of Ura dropout medium. The cells were harvested at 3,000 × g for 5 min, transferred to 1 liter of YPG (containing 2%galactose and 1%raffinose instead of glucose), and grown at 30 °C for 8 h. The Rab1A was then purified from the yeast as described previously for Rab3A (30Hutt D.M. Da-Silva L.F. Chang L.H. Prosser D.C. Ngsee J.K. J. Biol. Chem. 2000; 275: 18511-18519Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), except that phosphate-buffered saline was used instead of Tris-HCl and the proteins were eluted from the resin with 50 mm EDTA. The purified Rab3A and Rab1A were cross-linked to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech AB) as described by the manufacturer. Various domains of HA-tagged wild-type PRA1 and PRA2 were PCR amplified using pIRES/HA-PRA1 and pIRES/HA-PRA2 as DNA templates. Two rounds of PCR were used to generate the chimeras. The first round of PCR generated the individual PRA1 or PRA2 fragments, which served as templates for the second round to create the different chimeras. The PCR products were digested with ClaI and EcoRI and subcloned into the same restriction sites of the mammalian expression vector pIRESpuro (CLONTECH). The carboxyl regions of both PRA1 and PRA2 were PCR-amplified using the Bluescript KSII(−)/HA-PRA1 and KSII(−)/HA-PRA2 as DNA templates. PCR products were gel purified and subcloned into the GFP-containing vector, pEGFP-C1 (CLONTECH) as XhoI andBamHI fragments such that the GFP was fused to the amino terminus of the PCR product. All plasmids were sequenced to confirm the expected mutations. The binding of Rab1A and Rab3A to PRA1 and PRA2, in the presence of GDPβS or GTPγS was done using purified yeast or bacterially expressed proteins in a pull-down assay. A typical binding assay contained 20 μl of 50%bead slurry cross-linked with Rab1A or Rab3A at 2 pmol/μl. This was incubated for 1 h at 4 °C with 10, 15, and 20 pmol of recombinant PRA1 or PRA2 in a total volume of 250 μl of 25 mm Tris-HCl, pH 7.5, 125 mm KCl, 0.005%Triton X-100, 10%glycerol, 0.5 mm MgCl2, and 250 μm GDPβS or GTPγS (Roche Molecular Biochemicals). Controls were also done using Sepharose beads with no cross-linked Rab. The beads were then washed three times with 1 ml of ice-cold wash buffer (25 mmTris-HCl, pH 7.5, 125 mm KCl, and 0.005%Triton X-100, 10%glycerol). Denaturing loading buffer was added to the beads and proteins were subjected to Western immunoblot analysis. PRA1 and PRA2 were detected using anti-HA antibodies (Roche). All plasmid constructs were transfected into Chinese hamster ovary (CHO) cells using LipofectAMINE (Life Technologies). CHO cells were seeded at 1 × 105 on 12-mm diameter coverslips overnight and transfected with 0.5 μg of DNA in 1.5 μl of LipofectAMINE. The cells were fixed after 36–48 h in 4%paraformaldehyde (EM Sciences) in phosphate-buffered saline for 30–60 min and washed with 100 mm glycine in phosphate-buffered saline. Cells were incubated with blocking buffer (1%bovine serum albumin, 2%normal goat serum, and 0.4%saponin in phosphate-buffered saline) for 30–60 min at room temperature. Mouse monoclonal anti-HA antibodies (Roche Molecular Biochemicals) were diluted in blocking buffer and incubated with the cells for 1 to 2 h at room temperature. Cells were then washed with 100 mmglycine in phosphate-buffered saline and Alexa 488- or Alexa 568-labeled secondary antibodies (Molecular Probes) were used to detect the bound primary antibodies. Rabbit anti-calnexin (Stressgen) and anti-mannosidase-II (kindly provided by Dr. M. G. Farquhar) were used together with the monoclonal anti-HA in certain cases. Coverlips were mounted with Slow Fade anti-quench solution (Molecular Probes) and confocal laser microscopy (Bio-Rad MRC-1024MP) was used to capture the images. The cells were fixed and stained to visualize the chimeric constructs; however, live cells were imaged for the GFP fusions. For subcellular fractionation, 100-mm plates of CHO cells were transfected with pIRESpuro/HA-PRA2 in LipofectAMINE. After 24–48 h, the cells were harvested and homogenized in 1 ml of 10 mmTris-HCl, pH 7.5, 150 mm NaCl (TBS) supplemented with 2 mm phenylmethylsulfonyl fluoride. The crude homogenate was spun at 5,000 × g for 10 min to remove the cell debris, and the resulting supernatant was subjected to centrifugation at 100,000 × g for 1 h to yield the high speed cytosolic and intracellular membrane fractions. To further characterize the membrane-associated PRA2, the high speed membrane fractions were resuspended in 0.1 ml of TBS and extracted with 20 volumes of 0.1m sodium carbonate, pH 11.5, at 4 °C for 30–60 min. The membranes were then collected by centrifugation at 100,000 ×g for 1 h (4 °C). For Triton X-114 extraction, the resuspended membranes were solubilized in 1%Triton X-114 at 4 °C for 30–60 min, and induced to phase partition at 37 °C for 10–15 min followed by centrifugation at 10,000 × g for 5 min at room temperature. The resulting detergent phase was back extracted three times with 10 volumes of ice-cold TBS at 4 °C for 10 min, and phase partitioned as before. The resulting supernatants were pooled and precipitated with 10%trichloroacetic acid. Proteins involved in vesicle transport often exist as multiple isoforms with each mediating specific membrane trafficking steps. A BLAST search of the mouse and rat EST data bases with the conserved region of rat PRA1 revealed a number of overlapping clones. Merging the sequences from two of the longest mouse clones (accession numbers AW519501 and AA051031) revealed an open reading frame of 564 nucleotides. Oligonucleotides spanning the initiation and termination codons were used to PCR amplify rat and mouse brain cDNA libraries. Both gave a PCR product of the expected size, and were subsequently subcloned into a HA-tagged Bluescript vector for DNA sequence analysis. The open reading frame of the rat PRA2 contained 188 amino acids with a predicted molecular mass of 21.5 kDa. As with PRA1, PRA2 also contained two extensive hydrophobic domains, of 36 and 35 residues spanning amino acids 47 to 82 and 101 to 135, respectively. Comparison of the two rat PRA sequences revealed an overall amino acid identity of 26%and a similarity of 35%(Fig. 1). The most conserved domain was located at residues 35 to 47, immediately amino-terminal to the first hydrophobic segment of the two proteins. One notable difference between the two proteins was the length of the amino- and COOH-terminal domains flanking the two hydrophobic segments. PRA2 has a shorter amino-terminal domain of 46 residues compared with 77 in PRA1, but a longer COOH-terminal domain of 53 residues compared with 21 in PRA1. The PRA2 COOH-terminal domain has a cluster of basic residues in contrast to PRA1 which has a number of acidic residues. Moreover, this COOH-terminal domain of PRA2 has a weak coiled-coil conformation, which might play a role in protein-protein interaction. A search of the data base revealed that PRA2 is identical to the rat JWA (accession number AAF60354) and highly similar to the human JM4 (accession number NP 009144) clones. Northern blot analysis showed that PRA2 was encoded by a single 1.5–1.8-kilobase transcript and broadly expressed in most tissues (Fig. 2). The message was most abundant in the heart and brain. This expression pattern was similar to that of PRA1 (29Martincic I. Peralta M.E. Ngsee J.K. J. Biol. Chem. 1997; 272: 26991-26998Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) with one exception: there was a lower level of expression of PRA2 in the testis whereas PRA1 was highly expressed. This broad expression pattern suggests that PRA2 might also be involved in membrane trafficking events in all tissues. In both PRA1 and PRA2, the testis transcript appeared larger than that from other tissues. The reason for this remained unknown. Since PRA1 can bind both GDP- and GTP-bound Rab, we examined the interaction of PRA2 with the Rab GTPases. In the yeast two-hybrid system, PRA2 showed a positive β-galactosidase reaction when tested against the wild-type and GTPase mutant Rab1A and Rab3A (data not shown). As with PRA1, the interaction was abolished when the double Cys prenylation motif of Rab was deleted, suggesting that prenylation is required for interaction. To confirm this interaction, we performed an in vitro binding assay using recombinant Rab1A and Rab3A purified as a 6xHis-tagged fusion protein from the yeast, Saccharomyces cerevisiae, and covalently linked to CNBr-activated Sepharose. The proteins were pre-loaded with GDP, GTP, or maintained in the nucleotide-free state (in the presence of EDTA). Increasing amounts of recombinant HA-tagged PRA1 or PRA2 was added to the beads at 4 °C with the bound proteins recovered and analyzed by Western immunoblot. As shown in Fig.3 A, PRA2 was recovered with the immobilized Rab1A and Rab3A but not with the control Sepahrose beads. There was a slight increase in the amount of PRA2 recovered with immobilized Rab3A compared with Rab1A. PRA2 showed a slightly higher affinity for GTP-bound Rab but was clearly recovered with the GDP-bound as well as guanine nucleotide-free state of both Rab GTPases. Under the same conditions, PRA1 also showed a slight preference for Rab3A over Rab1A (Fig. 3 B). There was also a slightly higher affinity for the GTP-bound Rab followed by guanine nucleotide-free and GDP-bound forms. Thus, we conclude that both PRA1 and PRA2 can interact with, at least, Rab1A and Rab3A in the guanine nucleotide-bound and free states. To determine the cellular distribution, we subcloned the HA-tagged PRA2 into the bicistronic expression vector pIRESpuro, transfected it into CHO cells, and performed a subcellular fractionation analysis. As with PRA1, the protein fractionated with both the high speed supernatant and membranes (Fig. 4). When the membrane fraction was extracted with alkaline carbonate buffer, some of the membrane-bound protein appeared in the soluble fraction similar to the behavior of PRA1 and suggested peripheral association with the membrane. However, the membrane-bound PRA2 partitioned exclusively with the detergent phase when subjected to Triton X-114 extraction and phase separation. In contrast, a significant portion of the membrane-bound PRA1 remained with the aqueous phase in the Triton X-114 extraction. Thus, both PRA1 and PRA2 are highly hydrophobic proteins tightly associated with the membrane but may also appear in the cytosol. When transfected into CHO cells, PRA2 has a striking reticular staining pattern reminiscent of the ER (Fig.5 A). Indeed, there was extensive co-localization with calnexin, a known ER marker. We observed little, if any, co-localization with mannosidase II, a Golgi membrane marker. In contrast, PRA1 was found exclusively associated with the Golgi complex with extensive co-localization with mannosidase II (Fig.5, C and D). Thus, the two PRA isoforms are localized to distinct intracellular compartments with PRA2 restricted to the ER and PRA1 confined to the Golgi complex (30Hutt D.M. Da-Silva L.F. Chang L.H. Prosser D.C. Ngsee J.K. J. Biol. Chem. 2000; 275: 18511-18519Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This distinct intracellular localization implies that PRA1 and PRA2 may contain targeting signals that direct the protein to the appropriate intracellular compartment. It also implies that each PRA might only interact with a subset of Rab in the cell even though both are capable of binding multiple Rab isoforms in either the GDP- or GTP-bound state. To define the organelle-specific targeting signal, we constructed amino-terminal HA-tagged chimeras of PRA1 and PRA2, and determined their cellular localization in transfected CHO cells. Based on the structural features, we divided the two proteins into three separate domains: amino-terminal domain (Domain A), the two hydrophobic segments plus the intervening hydrophilic loop (Domain B), and the charged COOH-terminal domain (Domain C). Hybrid oligonucleotides spanning these domains were used to generate the chimeras by PCR using either HA-tagged PRA1 or PRA2 as the template. As shown in Fig.6, chimera 1A/2BC, for example, contained Domain A of PRA1 fused to Domains B and C of PRA2. Likewise, chimera 2A/1B/2C contained Domain A of PRA2 fused to Domain B of PRA1 followed by Domain C of PRA2, and so on. The various chimeras were subcloned into the pIRESpuro vector and sequenced to confirm the expected mutations. Two days after transfection, the CHO cells were stained with anti-HA and costained with either anti-mannosidase II or anti-calnexin to identify the Golgi and ER, respectively. As shown in Fig.7, the COOH-terminal domain of PRA2 targeted the chimera to the ER whether it contained only the amino-terminal Domain A of PRA1 (1A/2BC in Fig. 7 A and6 D), both Domains A and B of PRA1 (1AB/2C in Fig. 7,B and E), or only Domain B of PRA1 (2A/1B/2C in Fig. 7, C and F). Similarly, Domain C of PRA1 targeted the chimera to the Golgi complex whether it contained only the amino-terminal Domain A of PRA2 (2A/1BC in Fig. 7, G andJ), both Domains A and B of PRA2 (2AB/1C in Fig. 7,H and K), or only Domain B of PRA2 (1A/2B/1C in Fig. 7, I and L). Thus, we concluded that the COOH-terminal domain of PRA1 and PRA2 contained the localization signal that either targeted or caused the protein to be retained at the proper membrane compartment. The amino-terminal Domain A and the two hydrophobic segments in Domain B of the two proteins were interchangeable. For PRA1, its normal localization to the Golgi complex was not altered by the presence of either Domain A (2A/1BC) or Domain B (1A/2B/1C) of PRA2. Likewise, the normal position of PRA2 at the ER was not altered by the presence of either the amino-terminal Domain A (1A/2BC) or Domain B (2A/1B/2C) of PRA1.Figure 7Cellular localization of PRA1 and PRA2 chimeras. The transfected CHO were stained with anti-HA (panels A, C, E, G, I, and K) and with either calnexin (panels B, D, and F) or mannosidase II (panels H, J, and L). The cells were transfected with chimera 1A/2BC (panels A and B), 1AB/2C (panels C and D), 2A/1B/2C (panels Eand F), 2A/1BC (panels G and H), 2AB/1C (panels I and J), and 1A/2B/1C (panels K and L).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the COOH-terminal domain was indeed responsible for targeting the protein to the appropriate membrane compartment, we constructed chimeras containing the full-length or only Domain C of PRA1 and PRA2 fused to the carboxyl terminus of GFP. When transfected into CHO cells, the full-length PRA2 (Fig.8 B) and PRA1 (Fig.8 C) showed a bright fluorescent signal in the ER and Golgi complex, respectively. Thus, the full-length protein was able to direct a cytosolic GFP (Fig. 8 A) to a specific membrane organelle. Domain C of PRA2-(132–188) was able to partially target GFP to intracellular membranes where a distinctive punctate staining pattern throughout the cell was clearly evident (Fig. 8 D). However, membrane targeting by this domain was inefficient as a significant amount of florescent signal remained in the cytosol. Since this domain of PRA2 contained 57 amino acids, we performed a limited deletion to further define the boundaries of the localization signal. Further deletion of 17 residues in GFP-PRA2-(148–188) or 33 residues in GFP-PRA2-(164–188) completely abrogated this punctate staining pattern (Fig. 8, F and H). Thus, the minimal localization signal must either be contained within the first 17 amino acids immediately COOH-terminal to the second hydrophobic segment or required domains in addition to that contained within Domain C. In contrast to PRA2, GFP fusion of Domain C of PRA1-(162–185), which contained only 24 amino acids, cannot target GFP to intracellular membranes (Fig.8 E). Neither can a further addition of 5 hydrophobic residues in GFP-PRA1-(157–185). When combined with the previous observation, these results suggest that the COOH-terminal domain of PRA2 can partially target proteins to the membrane and that transport of PRA1 to the Golgi complex required additional domains such as ones that can functionally interact with Rab or VAMP2. One striking feature of the COOH-terminal domain of the PRA isoforms is their overall charge. PRA1 contained a number of acidic residues whereas the first 15 amino acids of Domain C in PRA2 contained numerous basic residues. The carboxyl terminus of PRA1 contained an additional glutamate immediately after the consensus DXE motif (residues 176–178) involved in the exit of membrane proteins from the ER (35Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (397) Google Scholar, 36Nishimura N. Bannykh S. Slabough S. Matteson J. Altschuler Y. Hahn K. Balch W.E. J. Biol. Chem. 1999; 274: 15937-15946Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To test whether this DXEE motif might constitute the sorting signal of PRA1, we mutated these