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Signal Sequences Specify the Targeting Route to the Endoplasmic Reticulum Membrane Davis T.W

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Signal Sequences Specify the Targeting Route to the Endoplasmic Reticulum Membrane Davis T.W Signal Sequences Specify the Targeting Route to the Endoplasmic Reticulum Membrane Davis T.W. Ng, Jeremy D. Brown, and Peter Walter Department of Biochemistry and Biophysics, University of California, School of Medicine, San Francisco, California 94143-0448 Abstract. In the yeast Saccharomyces cerevisiae, only a dent preproteins in vivo and in vitro, whereas SRP- subset of preproteins that are translocated across the dependent preproteins were unaffected. Based on this E R membrane require the function of the signal recog- analysis, preproteins fall into three distinct classes: SRP nition particle (SRP), suggesting that an alternative, dependent, SRP independent, and those that can use SRP-independent pathway must exist (Hann, B.C., and both pathways. Pathway specificity is conferred by the P. Walter. 1991. Cell. 67:131-144). We have established hydrophobic core of signal sequences. Our studies show that the two targeting pathways function in parallel. a previously unrecognized diversity in ER-directed sig- Mutant alleles of SEC62 and SEC63 were isolated that nal sequences, that carry structural information that specifically impaired the translocation of SRP-indepen- serves to identify the route taken. Downloaded from HE first steps of the eukaryotic secretory pathway absence of SRP and SRP receptor. Although the loss of include targeting and translocation of nascent pro- SRP function results in translocation defects for a number T teins across the ER membrane. In vitro analyses us- of ER-targeted proteins, the extent of the defect is vari- www.jcb.org ing components largely derived from mammalian sources able from one protein to another: at one extreme, translo- (for reviews see Walter and Johnson, 1994; Rapoport et cation of the vacuolar protease dipeptidyl aminopeptidase al., 1996) have revealed that targeting is catalyzed by the B (DPAP B) is severely affected; at the other extreme, signal recognition particle (SRP) 1 and its membrane-local- carboxypeptidase Y (CPY) is unaffected (Hann and Walter, on March 17, 2006 ized receptor. The translocation events are carried out by 1991). Furthermore, cells lacking SRP function "adapt" a multi-subunit membrane protein complex, the translocon over time and thereby gain the ability to translocate all or Sec61p complex (G6rlich and Rapoport, 1993). Mecha- proteins that have been examined so far (Ogg et al., 1992). nistically, translocation is cotranslational: SRP binds signal From these observations, it was hypothesized that an alter- sequences as they emerge from the ribosome, pauses native or salvage pathway must exist to translocate pro- translation, and targets the nascent chain/ribosome com- teins in the absence of SRP function. plexes to the ER membrane where they are docked onto In contrast to higher eukaryotes, components involved the translocon. in a posttranslational mode of translocation have been A posttranslational mode of translocation has also been identified in yeast whose functional equivalents in other reported for mammalian systems, but neither the mecha- organisms have yet to be characterized. These include nism nor components involved have been characterized Sec62p, Sec63p, Sec71p, and Sec72p (Deshaies and Schek- (Schlenstedt et al., 1990). These observations suggest that man, 1989; Rothblatt et al., 1989; Green et al., 1992; Feld- SRP-mediated targeting is not the only route through helm et al., 1993; Feldheim and Schekman, 1994) which form which proteins enter the secretory pathway. a complex (Sec62/63p complex) at the ER membrane. This Analyses of protein translocation across the ER mem- complex, together with the Sec61p complex (the mem- brane in budding yeast have reinforced the view that SRP, brane translocon), and Kar2p (BiP) associated with the SRP receptor, and translocon components are conserved ER lumen mediate translocation. Using in vitro reconsti- among all eukaryotic cells (Hann et al., 1989; G6rlich et tution assays, these were the only membrane constituents al., 1992; Ogg et al., 1992; Stirling et al., 1992; Brown et al., required to support a posttranslational mode of transloca- 1994). Surprisingly, however, yeast cells are viable in the tion into artificial proteoliposomes (Brodsky and Schekman, 1993; Panzner et al., 1995). Interestingly, some mutations Address all correspondence to Dr. Davis T.W. Ng, Department of Bio- in genes encoding these proteins, as with SRP mutants, chemistry and Biophysics, University of California School of Medicine, cause variable defects in translocation efficiency depend- San Francisco, CA. Tel.: (415) 476-5017. Fax: (415) 476-5233. E-mail: ing on the substrate (Deshaies and Schekman, 1989; Feld- [email protected] helm and Schekman, 1994). The significance of these ob- 1. Abbreviations used in this paper: CPY, carboxypeptidase Y; DPAP B, servations, however, was unclear. Because only a small dipeptidyl aminopeptidase B; SRP, signal recognition particle. number of substrates have been examined in these sys- © The Rockefeller University Press, 0021-9525/96/07/269/10 $2.00 The Journal of Cell Biology, Volume 134, Number 2, July 1996 269-278 269 terns, it was not known what role the Sec62/63p complex Clal fragment of the PCR product encoding the amino-terminal portion plays in the overall trafficking of secretory pathway pro- of the fusion protein was subcloned into pDN317 to create pDN321. (c) ct-F reporter protein: Using the MFal gene as the template, a fragment teins in yeast. bearing a BamHl site upstream of the initiator codon and an Ncol site up- To examine the role of multiple translocation pathways stream of the terminator was generated by PCR. Along with an HA (defined here to include both targeting and membrane epitope tag bearing Ncol/Xbal fragment, it was inserted into pDN201 gen- translocation steps), we devised a genetic strategy to select erating the plasmid pDN229, pDN229 encodes prepro-a-factor with a car- for mutants specifically defective in an SRP-independent boxyl-terminal HA epitope tag. (d) Ds-a-F reporter protein: Using DAP2 as the template, the first PCR step used the D~ 5' primer and a 3' primer route to the ER. From this selection, we obtained mutants encoding the designed DPAP B/c~-F fusion junction (5'-GAATTFGTGC- that severely impair the translocation of substrates unaf- CDGTITCTATACFIWITAGCAAC-3') to generate the 5' megaprimer fected in cells lacking SRP function while leaving SRP- for the second step. The PCR second step and subsequent cloning was as mediated translocation intact. By combining genetic and performed as for the a-F construct but using the megaprimer as the 5' primer. The resulting expression vector was designated pDN280. (e) DHc- biochemical approaches, we have dissected two pathways: a-F reporter protein: Using the DN-a-F coding sequences as a template, a one dependent and the other independent of SRP. Using 5' PCR primer encoding the first two residues of prepro-a-factor fused to this system to examine a wide variety of endogenous sub- hydrophobic core sequences of DPAP B was used to amplify the gene. strates, we show that both pathways are required to pro- The amplified fragment inserted into pDN201 generated the plasmid cess the full complement of secretory pathway proteins. pDN314. For in vitro studies, coding sequences were inserted into BamHl and Analysis of recombinant chimeric substrates demonstrates XbaI sites in pSP65 generating plasmids pJD75 (DN-ct-F) and pJD82 that the hydrophobic core of signal sequences serves as an (Dnc-a-F). Myc epitope-tagged PH08 was inserted into pSP65 as a "address" for entry into either pathway. This is surprising BamHI-Pstl fragment from pTPPP creating plasmid pJD76. PRCI coding since signal sequences show no primary structure conser- sequences were amplified by PCR from yeast genomic DNA and inserted between the Smal and Hindlll sites of pSP65 creating plasmid pJD77. vation suggesting that they have little specific information content (von Heijne, 1985; Kaiser et al., 1987). Thus, our studies show a previously unrecognized diversity in ER- Genetic Selection directed signal sequences, which carry specific structural DNY53 cells were mutagenized with UV light irradiation at rates resulting information that serves to identify the route taken. in 30 and 57% lethality. 1.4 × l0 T cells irradiated to 30% lethality and 3.4 Downloaded from × 107 cells irradiated to 57% lethality were plated out in various densities on SC media lacking uracil and incubated at 22°C. Over the course of 5 d Materials and Methods after plating, a total of 240 Ura+ colonies were picked and restreaked onto selective media. Only the larger colonies were picked; many colonies that grew slowly because they were sick or only slightly Ura+ were not in- Strains Used in this Study cluded in this first group. Of the 240 isolates, 59 were eliminated due to slow growth on SC-uracil media. The rest were crossed to a wild-type YJL183 (MA Ta, ura3A99, leu2A1, trp1A99, ade2-1Ol°~hre), YJL184 (MATa, www.jcb.org strain (W303) to form diploids. The dominant or recessive nature of the YJL183 background) from Dr. J. Li, University of California, San Fran- mutants was determined by assessing the ability to confer uracil prototro- cisco; W303 (MATt~, leu2-3-112, his3-11, trpl-1, ura3-1, canl-lO0, ade2-1); phy in the heterozygous diploids. The growth of diploid cells on selective DNY53 (YJL183, pDN106), DNY54 (YJL184, pDN106), DNY66 media indicated three mutant categories: (1) dominant: growth indistin- (DNY54, sec63-201), DNY70 (DNY54, sec62-101), DNY116 (DNY54, guishable from haploid mutant; (2) semi-dominant: some growth but on March 17, 2006 sec61-101) from this study; SOY162 (W303, MATct, srplO2.'.'HIS3) [SRI3A] worse than the haploid mutant; (3) recessive: growth identical to wild-type from Dr. S. Ogg, University of California, San Francisco); PS886 (MATala, ceils carrying the reporter. 95 mutants fell in the first two categories and trpl, leu2, ura3-52, pep4-3, prb, prc, barl::LEU2/BAR1) from Dr. P. were not examined further.
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