Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.     

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75

The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.     

Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins.

In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane.     

Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.     

The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains.

The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism.     

Identification of the protein storage vacuole and protein targeting to the vacuole in leaf cells of three plant species

Protein storage vacuoles (PSVs) are specialized vacuoles devoted to the accumulation of large amounts of protein in the storage tissues of plants. In this study, we investigated the presence of the storage vacuole and protein trafficking to the compartment in cells of tobacco (Nicotiana tabacum), common bean (Phaseolus vulgaris), and Arabidopsis leaf tissue. When we expressed phaseolin, the major storage protein of common bean, or an epitope-tagged version of alpha-tonoplast intrinsic protein (alpha-TIP, a tonoplast aquaporin of PSV), in protoplasts derived from leaf tissues, these proteins were targeted to a compartment ranging in size from 2 to 5 microm in all three plant species. Most Arabidopsis leaf cells have one of these organelles. In contrast, from one to five these organelles occurred in bean and tobacco leaf cells. Also, endogenous alpha-TIP is localized in a similar compartment in untransformed leaf cells of common bean and is colocalized with transiently expressed epitope-tagged alpha-TIP. In Arabidopsis, phaseolin contained N-glycans modified by Golgi enzymes and its traffic was sensitive to brefeldin A. However, trafficking of alpha-TIP was insensitive to brefeldin A treatment and was not affected by the dominant-negative mutant of AtRab1. In addition, a modified alpha-TIP with an insertion of an N-glycosylation site has the endoplasmic reticulum-type glycans. Finally, the early step of phaseolin traffic, from the endoplasmic reticulum to the Golgi complex, required the activity of the small GTPase Sar1p, a key component of coat protein complex II-coated vesicles, independent of the presence of the vacuolar sorting signal in phaseolin. Based on these results, we propose that the proteins we analyzed are targeted to the PSV or equivalent organelle in leaf cells and that proteins can be transported to the PSV by two different pathways, the Golgi-dependent and Golgi-independent pathways, depending on the individual cargo proteins.     

Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site.

The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha-TIP showed that the TIPs are differentially expressed.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.     

Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes

Members of the major intrinsic protein (MIP) family, described in plants as water-selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non-electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free-flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped-flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 +/- 9.6 x 10(-6) and 1.0 +/- 0.3 x 10(-6) cm sec-1, respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel-mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt-TIPa) was isolated from tobacco cells. Immunodetection of Nt-TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt-TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Nt-TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.     

A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium

Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1] [2]. It also interacts with the cytoplasmic tail of beta integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4] [5] [6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece.     

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

Targeting of P-selectin to two regulated secretory organelles in PC12 cells

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P- selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.     

An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF).

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.     

The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin''s cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

Internalization of proprotein convertase PC7 from plasma membrane is mediated by a novel motif

Proprotein convertase 7 (PC7) is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network, but a small fraction is found at the cell surface. So far, no sorting signals for membrane trafficking have been identified in PC7. In this study, we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles. After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor, Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated clathrin-coated vesicles. To determine the internalization motif, constructs were generated in which parts of the N and C terminus of the cytoplasmic tail of PC7 were deleted, and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between Ala(713) and Cys(726) in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for trans-Golgi network localization. Individual amino acids in this region were substituted with alanine, which identified Pro, Leu, and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail, which contains the three crucial amino acids PLC.     

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.     

PDZ domain protein GIPC interacts with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-related protein-1).

Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation. Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains. To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library. Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein. The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein. Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC. Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state. Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region. Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes.     

Sorting signals within the Saccharomyces cerevisiae sporulation-specific dityrosine transporter, Dtr1p, C terminus promote Golgi-to-prospore membrane transport

During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.