Bipartite Signals Mediate Subcellular Targeting of Tail-anchored Membrane Proteins in Saccharomyces cerevisiae

Tail-anchored proteins have an NH2-terminal cytosolic domain anchored to intracellular membranes by a single, COOH-terminal, transmembrane segment. Sequence analysis identified 55 tail-anchored proteins inSaccharomyces cerevisiae, with several novel proteins, including Prm3, which we find is required for karyogamy and is tail-anchored in the nuclear envelope. A total of six tail-anchored proteins are present in the mitochondrial outer membrane and have relatively hydrophilic transmembrane segments that serve as targeting signals. The rest, by far the majority, localize via a bipartite system of signals: uniformly hydrophobic tail anchors are first inserted into the endoplasmic reticulum, and additional segments within the cytosolic domain of each protein can dictate subsequent sorting to a precise destination within the cell. Tail-anchored proteins have an NH2-terminal cytosolic domain anchored to intracellular membranes by a single, COOH-terminal, transmembrane segment. Sequence analysis identified 55 tail-anchored proteins inSaccharomyces cerevisiae, with several novel proteins, including Prm3, which we find is required for karyogamy and is tail-anchored in the nuclear envelope. A total of six tail-anchored proteins are present in the mitochondrial outer membrane and have relatively hydrophilic transmembrane segments that serve as targeting signals. The rest, by far the majority, localize via a bipartite system of signals: uniformly hydrophobic tail anchors are first inserted into the endoplasmic reticulum, and additional segments within the cytosolic domain of each protein can dictate subsequent sorting to a precise destination within the cell. soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors 4′,6-diamidino-2-phenylindole nuclear localization sequence GTP exchange factor GTP-activating protein Tail-anchored proteins have a single transmembrane segment at their carboxyl terminus, and many of the proteins that mediate subcellular traffic and programmed cell death are tail-anchored into select membranes of eukaryotic cells (1Wattenberg B. Lithgow T. Traffic. 2001; 2: 66-71Google Scholar). In the Bcl-2 family of proteins, key regulators of the programmed cell death pathway in animal cells, 12 of the 16 known family members are tail-anchored to either the mitochondrial outer membrane or endoplasmic reticulum, and their membrane location is critical for function (2Antonsson B. Cell Tissue Res. 2001; 306: 347-361Google Scholar). TheSNAP-receptors (SNAREs)1 are a family of proteins essential for intracellular membrane fusion, and 19 of the 23 SNAREs in yeast are tail-anchored proteins. Membrane fusion absolutely requires the participation of SNAREs in both the donor and acceptor membrane, and each of the known SNAREs has a restricted location at a defined membrane compartment of the endomembrane system (3Pelham H.R. Exp. Cell Res. 1999; 247: 1-8Google Scholar, 4Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 98-106Google Scholar). Tail-anchored proteins fold co-translationally and the single hydrophobic segment at their carboxyl terminus allows for post-translational insertion into membranes (5Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapport T.A. EMBO J. 1995; 14: 217-223Google Scholar, 6Federov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Google Scholar). Once membrane is inserted, the amino-terminal domain of the protein is displayed in the cytosol. An elegant study on the tail-anchored SNARE synaptobrevin-1/VAMP-1a found that the protein is inserted into the endoplasmic reticulum and subsequently sorted to presynaptic vesicles (5Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapport T.A. EMBO J. 1995; 14: 217-223Google Scholar). Cytochrome b 5, another tail-anchored protein, is inserted into the membrane of the endoplasmic reticulum and maintained there despite some escape to, and retrieval from, thecis-Golgi cisternae (7Pedrazzini E. Villa A. Longhi R. Bulbarelli A. Borgese N. J. Cell Biol. 2000; 148: 899-914Google Scholar). But tail-anchored proteins are also located in the mitochondrial outer membrane, and the precise signal that distinguishes these from those targeted to the endoplasmic reticulum is still not clear. Deletion mutagenesis has shown the signal is contained within the tail segment, and in the few different model proteins examined to date critical determinants have been either the presence of charged residues or in some cases the number of hydrophobic residues (8Mitoma J. Ito A. J. Biochem. (Tokyo). 1991; 111: 20-24Google Scholar, 9Vergeres G. Waskell L. J. Biol. Chem. 1992; 267: 12583-12591Google Scholar, 10Borgese N. Gazzoni I. Barberi M. Colombo S. Pedrazzini E. Mol. Biol. Cell. 2001; 12: 2482-2496Google Scholar, 11Kuroda R. Ikenoue T. Honsho M. Tsujimoto S. Mitoma J.Y. Ito A. J. Biol. Chem. 1998; 273: 31097-31102Google Scholar, 12Isenmann S. Khew-Goodall Y. Gamble J. Vadas M. Wattenberg B.W. Mol. Biol. Cell. 1999; 9: 1649-1660Google Scholar, 13Egan B. Beilharz T. George R. Isenmann S. Gratzer S. Wattenberg B. Lithgow T. FEBS Lett. 1999; 451: 243-248Google Scholar, 14Allen R. Egan B. Gabriel K. Beilharz T. Lithgow T. FEBS Lett. 2002; 514: 347-350Google Scholar). An understanding of the precise targeting signals that direct the majority of tail-anchored proteins to the endoplasmic reticulum, but allow some to go exclusively to mitochondria, has been hampered by the relatively small number of model tail-anchored proteins that have been available for study. We applied several bioinformatic approaches to identify tail-anchored proteins encoded in the genome ofSaccharomyces cerevisiae. Fifteen novel tail-anchored proteins were discovered; we report here on the localization of the previously unrecognized tail-anchored proteins. Analysis of the targeting segments from the 55 tail-anchored proteins in yeast suggests a bipartite system of signals: hydrophobic character in the tail segment determines targeting to the endoplasmic reticulum instead of mitochondria and discrete sorting signals that then direct tail-anchored proteins to their correct subcellular destination. DNA fragments corresponding to each open reading frame were amplified by PCR using primers that generated one in-frame restriction site immediately preceding the start codon and another following the stop codon (oligonucleotide sequences available on request). PCR products were cloned behind GFP-S65T under the control of the MET25 promoter and expressed from a centromeric plasmid (13Egan B. Beilharz T. George R. Isenmann S. Gratzer S. Wattenberg B. Lithgow T. FEBS Lett. 1999; 451: 243-248Google Scholar). In semisynthetic (SD) media, expression from the plasmid is partially repressed. PCR-based mutagenesis was used to convert hydrophilic residues in the transmembrane segments of Tom22 and Fis1 to leucine residues. For the mutant described here, Fis1(4L), Gly136, Gly137, Gly141, and Ala142 were converted to leucine residues. Initial trials to visualize expression of the fusion proteins were made in the diploid strain JK9–3da/α (leu2–3,122/leu2–3,122, ura3–52/ura3–52, rme1/rme1 trp1/trp1, his4/his4 GAL+/GAL+, HMLa/HMLa). In three cases, this failed to give discernible fluorescence but expression of fusion proteins constructed from YBL100c, YFL046w, and YPL200c was possible using a strain defective in proteasome function (Matα, trp1, ura3, his, leu2, cim5–1; Ghislainet al. (38Ghislain M. Udvardy A. Mann C. Nature. 1993; 366: 358-362Google Scholar)). To generate yeast mutants lacking theFIS1 gene or the PRM3 gene, PCR-mediated gene disruption (15Wach A. Brachat A. Alberti-Segui C. Rebischung C. Philippsen P. Yeast. 1997; 13: 1065-1075Google Scholar) was employed with the plasmid p3xHA-His5 as template. For fluorescence microscopy, cells were visualized directly or after staining with Mitotracker (MitoTracker Red CM-H2X Ros) according to the standard protocol from Molecular Probes. All fluorescence images were captured using a Bio-Rad MRC1024 confocal scanning laser microscope mounted on a Zeiss Axioscop. For the mating studies, a Nikon fluorescence microscope with GFP and DAPI filter sets and a ×100 DIC (Nomarski) objective was used. In this case images were captured by a Micromax digital camera with Metamorph imaging software. In preparation for fluorescence microscopy cells were grown to mid-log phase at 25 °C in semisynthetic medium. In assays for nuclear transport, wild-type, rna1-1, orprp20-1 cells were transferred from 25 °C liquid culture to a 37 °C water bath for 30 min immediately prior to preparation for microscopy. Microsomal membrane fractions were prepared by differential centrifugation. Cells (50–100 OD600 units) were suspended in buffer 88 (250 mm sorbitol, 150 mm potassium acetate, 5 mm magnesium acetate, 0.5 mmphenylmethylsulfonyl fluoride, 1.2 μg/ml leupeptin, 0.75 μg/ml antipain, 0.25 μg/ml chymostatin, 1 μg/ml pepstatin, 50 mm HEPES, pH 6.8) and disrupted by two bursts (each of 2-min duration) in a mini-beadbeater-8 (Biospec products) using silica/zirconia beads. Cell debris was removed by centrifugation at 500 × g for 5 min. A crude membrane fraction was collected by centrifugation at 16,000 × g for 10 min. Membranes were extracted by resuspension in either 1% Triton X-100 or 100 mm Na2CO3 and incubated for 30 min on ice with intermittent vortexing. Soluble and insoluble proteins were separated by centrifugation at 100,000 ×g in a Beckman Airfuge. Published procedures were used for isolation of mitochondria and trypsin shaving, SDS-PAGE, and immunoblot analysis (16Beilharz T. Suzuki C. Lithgow T. J. Biol. Chem. 1998; 273: 35268-35272Google Scholar). Detailed comparative hydrophobicity analyses of the targeting sequences in each tail-anchored protein made use of the ProtParam site at (expasy.proteome.org.au/cgi-bin/protparam) using a window of 5 amino acids to scan through the predicted transmembrane segment according to the Kyte-Doolittle algorithm. In addition to the tail-anchored proteins known in yeast, sequence analysis revealed 15 open reading frames that could be expressed as GFP fusions that localize to discrete subcellular membranes. Fig.1 shows that Fis1 and YFL046w are mitochondrial proteins (Fig. 1 A), seven proteins localized generally to the endoplasmic reticulum membrane (Fig. 1 B), and four are localized to specific subdomains of the endoplasmic reticulum (Fig. 1 C). YLR238w and YOR324c are found in clusters within the bounds of the endoplasmic reticulum, and Prm3 is confined to the perinuclear (nuclear envelope) membrane. YPL206c, a protein showing sequence similarity to bacterial glycerophosphodiester phosphodiesterases, was found concentrated in lipid bodies, regions of endoplasmic reticulum specialized for lipid metabolism (Fig.1 C; Ref. 17Zweytick D. Athenstaedt K. Daum G. Biochim. Biophys. Acta. 2000; 1469: 101-120Google Scholar). Two proteins closely related in primary structure, YDL012c and YBR016w, are located in the plasma membrane (Fig. 1 D). Both YDL012c and YBR016w are localized more intensely in regions of new membrane synthesis, toward the emergent buds of dividing cells and also in the schmoo structure (Fig.1 E, arrowheads) of haploid cells in the presence of mating pheromone. Comparative sequence analysis of the carboxyl-terminal segments of the 41 tail-anchored proteins targeted to the endoplasmic reticulum (including the 17 proteins sorted to other membranes of the secretory pathway) revealed no obvious motifs in the primary structure that might serve as a common targeting signal. However, hydropathy analysis through the transmembrane segments suggests regions of high (>3.3 units) hydropathy score in each protein (Fig.2 A). Conversely, hydropathy analysis of the predicted transmembrane domain from Fis1 (Fig.2 B) and for YFL046w, Tom5, Tom6, Tom7, and Tom22 (data not shown) suggests this segment of each polypeptide is more amphipathic than for proteins targeted to the endoplasmic reticulum. Previous work has shown that the transmembrane segments of the translocase subunits Tom5, Tom6, Tom7, Tom22, and Fis1 are necessary and sufficient for targeting mitochondria (13Egan B. Beilharz T. George R. Isenmann S. Gratzer S. Wattenberg B. Lithgow T. FEBS Lett. 1999; 451: 243-248Google Scholar, 14Allen R. Egan B. Gabriel K. Beilharz T. Lithgow T. FEBS Lett. 2002; 514: 347-350Google Scholar,18Cao W. Douglas M.G. Biochem. Biophys. Res. Commun. 1996; 224: 457-461Google Scholar). 2T. Beilharz and T. Lithgow, unpublished observations. To test whether the character in the transmembrane segment of Fis1 distinguished it as a protein destined for mitochondria, site-directed mutagenesis was used to replace hydrophilic residues with leucines, such that the hydrophobicity approached that of proteins targeted to the endoplasmic reticulum (Fig. 2 B). When yeast cells expressing these mutant proteins as GFP fusions were analyzed by fluorescence microscopy, the Fis1 mutant, Fis1(L4), is targeted to the endoplasmic reticulum (Fig. 2 C). Similar results were found with mutations made in the transmembrane segment of Tom22 (data not shown). Fluorescence is sometimes also observed in the lumen of the vacuole, perhaps reflecting turnover of the inappropriately targeted proteins (Fig. 2 C, “V”). Tail-anchored proteins in the endoplasmic reticulum can display distinct patterns of localization. For example YOR324c is found in discrete clusters throughout the endoplasmic reticulum (Fig. 3 A), whereas Bos1 is a SNARE found sparingly in the endoplasmic reticulum but enriched in the Golgi (Fig. 3 B, arrows). The hydrophobic tail segments from each of these proteins are sufficient to target GFP to the endoplasmic reticulum, where it remains generally distributed (Fig. 3, C and D). The proteins are integrated into the membrane as judged by their resistance to extraction by sodium carbonate treatment of isolated membranes (Fig.3 E; data not shown). We identified a single protein, Prm3, whose distribution was restricted to the nuclear envelope and sought to understand whether its targeting and sorting were also mediated by bipartite signals. A series of deletion mutants were constructed (Fig.4 A) and tested for subcellular localization. Deletion of 91 amino acids, leaving only the carboxyl-terminal segment of Prm3 (GFP-Prm3Δ1–91), targets the GFP reporter to the endoplasmic reticulum (Fig. 4 B), which is continuous with the outer membrane of the nuclear envelope (19Prinz W.A. Grzyb L. Veenhuis M. Kahana J.A. Silver P.A. Rapoport T.A. J. Cell Biol. 2000; 150: 461-474Google Scholar). A second signal is present in Prm3(Δ1–68), causing the fusion to be localized specifically to the nuclear envelope (Fig. 4 C). Sequence analysis suggested a nuclearlocalization sequence (NLS) between Pro68 and Lys75. Point mutations in the putative NLS caused degradation of the full-length Prm3 fusion protein, and so to test whether the putative NLS is functional, we analyzed a soluble version, GFP-Prm3(Δ109–133). GFP-Prm3(Δ109–133) is localized exclusively within the nucleus, and confocal sectioning revealed it distributed throughout the nucleoplasm (Fig. 4 D). To be certain the information for nuclear localization is contained within the amino acid sequence P68GRVRKHK75, site-directed mutagenesis was used to convert Lys73 to Ala73, and the mutant protein GFP-Prm3(Δ109–133)* is compromised in import, with much of the protein located in the cytosol (Fig.4 E). If Prm3 has a bipartite targeting sequence, with the P68GRVRKHK75 truly acting as an NLS, the general nuclear import machinery of the cell should be required to maintain the full-length, membrane-embedded Prm3 within the nucleus. Ran is a small GTP-binding protein that plays a critical role in transport of proteins into the nucleus (20Chook Y.M. Blobel G. Curr. Opin. Struct. Biol. 2001; 11: 703-715Google Scholar, 21Kuersten S. Ohno M. Mattaj I.W. Trends Cell Biol. 2001; 11: 497-503Google Scholar). A gradient of Ran-GTP across the nuclear membrane is established by the differential localization of two enzymes: the Ran-GTPexchange factor (Ran-GEF) in the nucleus and the Ran-GTP-activating protein (Ran-GAP) in the cytosol, and mutations in either of these two enzymes leads to collapse of nuclear traffic. Temperature-sensitive mutants for Ran-GEF (prp20-1) and Ran-GAP (ran1-1) were transformed and the distribution of Prm3 measured by fluorescence microscopy. At the permissive temperature of 25 °C, the localization of Prm3 in the Ran-GEF mutants (Fig. 4 F) and the Ran-GAP mutants (Fig.4 G) is nuclear. However, after shift to the non-permissive temperature of 37 °C, Prm3 is found throughout the membranes of the endoplasmic reticulum, suggesting that without a functioning Ran cycle the protein cannot be sorted from the outer to inner membrane of the nuclear envelope. Since Prm3 is the first protein described in yeast that might be localized to the inner membrane of the nuclear envelope, we sought to determine its function by analysis of Δprm3 mutant cells. yeast cells of mating either with or with Δprm3 were and incubated on for then and with DAPI to visualize In nuclear DNA is by the the first diploid the of the two cells of the mating A). However, the that from Δprm3 × Δprm3 are to and nuclear remains distinct but at the where the through the whether staining for acid (Fig. 5 B) or protein C). as suggested by this microscopy, Prm3 is required for the fusion of nuclear then Δprm3 cells should haploid through subsequent To test this from mating those described in 5 were isolated by The from are in the of at each of the cells (Fig. 5 cannot with haploid cells of either mating and on (data not shown). the from Δprm3 × Δprm3 were exclusively the cells that from these have a single, of cannot be but can with cells of the mating (data not shown). The that from these a distinct with cells isolated from one of the mating and cells from the other of mating unpublished observations. has shown that the PRM3 gene is in to both M.G. P. J. Cell Biol. 2000; Scholar) and J. J. Biol. Chem. 2001; and both in karyogamy by Rev. Cell Biol. 1996; 12: Scholar)). Prm3 localization is on a NLS and the we Prm3 as the first of the karyogamy machinery to function in membrane fusion at the of the inner membrane of the nuclear envelope. In addition to the and localization of a of novel tail-anchored proteins, analysis of the sequence data by these proteins suggests a general model for the targeting of tail-anchored proteins to each intracellular membrane. analysis of the 41 tail-anchored proteins known to localize to membranes of the secretory the only common we could identify was the uniformly hydrophobic of the residues within the transmembrane segment. Previous on tail-anchored proteins have each suggested that the targeting information (8Mitoma J. Ito A. J. Biochem. (Tokyo). 1991; 111: 20-24Google Scholar, 9Vergeres G. Waskell L. J. Biol. Chem. 1992; 267: 12583-12591Google Scholar, 10Borgese N. Gazzoni I. Barberi M. Colombo S. Pedrazzini E. Mol. Biol. Cell. 2001; 12: 2482-2496Google Scholar, 11Kuroda R. Ikenoue T. Honsho M. Tsujimoto S. Mitoma J.Y. Ito A. J. Biol. Chem. 1998; 273: 31097-31102Google Scholar, 12Isenmann S. Khew-Goodall Y. Gamble J. Vadas M. Wattenberg B.W. Mol. Biol. Cell. 1999; 9: 1649-1660Google Scholar, 13Egan B. Beilharz T. George R. Isenmann S. Gratzer S. Wattenberg B. Lithgow T. FEBS Lett. 1999; 451: 243-248Google Scholar, 14Allen R. Egan B. Gabriel K. Beilharz T. Lithgow T. FEBS Lett. 2002; 514: 347-350Google Scholar). The hydrophobic tail segments of Prm3, and YOR324c are sufficient to target each of these proteins to the endoplasmic reticulum, and these fusions uniformly distributed through the perinuclear endoplasmic reticulum the outer membrane of the nuclear envelope) and endoplasmic reticulum. A segment of hydrophobic and is found in the six tail-anchored proteins targeted to the mitochondrial outer membrane. of these residues with leucines, to the hydrophobicity and perhaps other such as the of the it can targeting to The machinery protein insertion into the outer membrane P. N. FEBS Lett. 1993; Scholar, J. Biol. Chem. 1996; Scholar, L. Isenmann S. Wattenberg B.W. Biochem. J. 2000; as as the import of soluble proteins into mitochondria G. Nature. 1997; Scholar, W. Rev. Biochem. 1997; Scholar, W. T. N. Biochim. Biophys. Acta. 1999; Scholar, K. Lithgow T. Trends Biochem. 2001; Scholar). The structure of the suggests that it has only a on its into which the hydrophobic of the targeting sequences found to soluble proteins targeted to mitochondria Y. T. T. K. S. T. D. Cell. 2000; Scholar, T. T. Y. T. T. D. J. Mol. Biol. 2001; 306: Scholar). has been as the import for tail-anchored proteins, and it be of to determine the of the the targeting segments of tail-anchored proteins. In to insertion of tail-anchored proteins into mitochondria, the of the machinery that tail-anchored protein insertion into the endoplasmic reticulum is still al. (5Kutay U. Ahnert-Hilger G. Hartmann E. Wiedenmann B. Rapport T.A. EMBO J. 1995; 14: 217-223Google Scholar) suggested that the general post-translational machinery might mediate insertion of tail-anchored proteins the it is to tail-anchored proteins with hydrophobic a of leucine residues was a for in insertion into membrane vesicles from the endoplasmic reticulum P. E. U. Rapoport T.A. G. J. Biol. Chem. 1996; and the tail segment GFP to the endoplasmic reticulum expressed in yeast regions in the cytosolic domain of its subsequent transport from the endoplasmic reticulum to the presynaptic mediated by distinct regions in the cytosolic are also required for Bos1 to the Prm3 targeting to the nuclear envelope, and YOR324c in of the endoplasmic reticulum. two targeting to the endoplasmic reticulum and subsequent sorting to the correct membrane, are mediated by bipartite targeting and sorting signals. For the Golgi and sorting be mediated through traffic. In the case of Prm3 and other proteins that might in the inner membrane of the nuclear envelope, we that this via the lipid of membrane present in the nuclear S. E. J. Cell 2000; Scholar, EMBO J. 2001; Scholar) that the outer and inner membrane and is by Ran and the that also the import of soluble nuclear proteins. We and for and and for critical on the and Wattenberg and for on the and critical throughout the of the