Abstract
We have cloned the gene for the resident luminal ER protein BiP from the fission yeast, Schizosaccharomyces pombe. The predicted protein product is equally divergent from the budding yeast and mammalian homologues. Disruption of the BiP gene in S. pombe is lethal and BiP mRNA levels are regulated by a variety of stresses including heat shock. Immunofluorescence of cells expressing an epitope-tagged BiP protein show it to be localized to the nuclear envelope, around the cell periphery and in a reticular structure through the cytoplasm. Unexpectedly, we find the BiP protein contains an N-linked glycosylation site which can be utilized. The C-terminal four amino acids of BiP are Ala-Asp-Glu-Leu, a new variant of the XDEL sequence found at the C-termini of luminal endoplasmic reticulum proteins. To determine whether this sequence acts as a sorting signal in S.pombe we expressed an acid phosphatase fusion protein extended at its C-terminus with the amino acids ADEL. Analysis of the sorting of this fusion protein indicates that the ADEL sequence is sufficient to cause the retention of proteins in the endoplasmic reticulum. The sequences DDEL, HDEL and KDEL can also direct ER-retention of acid phosphatase in S.pombe.
Full text
PDF![1583](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/fd7c9f48de43/emboj00089-0351.png)
![1584](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/7d2573caf0f6/emboj00089-0352.png)
![1585](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/98cb3e0fd697/emboj00089-0353.png)
![1586](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/d5d9c319d477/emboj00089-0354.png)
![1587](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/37c6403e3d15/emboj00089-0355.png)
![1588](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/bdc97aab9398/emboj00089-0356.png)
![1589](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/ea73d0979d04/emboj00089-0357.png)
![1590](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/d1d85ee2cc6d/emboj00089-0358.png)
![1591](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3ca0/556607/5f99d52b5873/emboj00089-0359.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blount P., Merlie J. P. BIP associates with newly synthesized subunits of the mouse muscle nicotinic receptor. J Cell Biol. 1991 Jun;113(5):1125–1132. doi: 10.1083/jcb.113.5.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chappell T. G., Warren G. A galactosyltransferase from the fission yeast Schizosaccharomyces pombe. J Cell Biol. 1989 Dec;109(6 Pt 1):2693–2702. doi: 10.1083/jcb.109.6.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dabora S. L., Sheetz M. P. The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell. 1988 Jul 1;54(1):27–35. doi: 10.1016/0092-8674(88)90176-6. [DOI] [PubMed] [Google Scholar]
- Dean N., Pelham H. R. Recycling of proteins from the Golgi compartment to the ER in yeast. J Cell Biol. 1990 Aug;111(2):369–377. doi: 10.1083/jcb.111.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fawell E., Hook S., Armstrong J. Nucleotide sequence of a gene encoding a YPT1-related protein from Schizosaccharomyces pombe. Nucleic Acids Res. 1989 Jun 12;17(11):4373–4373. doi: 10.1093/nar/17.11.4373. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fawell E., Hook S., Sweet D., Armstrong J. Novel YPT1-related genes from Schizosaccharomyces pombe. Nucleic Acids Res. 1990 Jul 25;18(14):4264–4264. doi: 10.1093/nar/18.14.4264. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
- Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
- Haas I. G., Wabl M. Immunoglobulin heavy chain binding protein. Nature. 1983 Nov 24;306(5941):387–389. doi: 10.1038/306387a0. [DOI] [PubMed] [Google Scholar]
- Hagan I. M., Hyams J. S. The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe. J Cell Sci. 1988 Mar;89(Pt 3):343–357. doi: 10.1242/jcs.89.3.343. [DOI] [PubMed] [Google Scholar]
- Haubruck H., Engelke U., Mertins P., Gallwitz D. Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue. EMBO J. 1990 Jun;9(6):1957–1962. doi: 10.1002/j.1460-2075.1990.tb08323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hengst L., Lehmeier T., Gallwitz D. The ryh1 gene in the fission yeast Schizosaccharomyces pombe encoding a GTP-binding protein related to ras, rho and ypt: structure, expression and identification of its human homologue. EMBO J. 1990 Jun;9(6):1949–1955. doi: 10.1002/j.1460-2075.1990.tb08322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kassenbrock C. K., Garcia P. D., Walter P., Kelly R. B. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature. 1988 May 5;333(6168):90–93. doi: 10.1038/333090a0. [DOI] [PubMed] [Google Scholar]
- Kozutsumi Y., Segal M., Normington K., Gething M. J., Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. doi: 10.1038/332462a0. [DOI] [PubMed] [Google Scholar]
- Kumar N., Syin C. A., Carter R., Quakyi I., Miller L. H. Plasmodium falciparum gene encoding a protein similar to the 78-kDa rat glucose-regulated stress protein. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6277–6281. doi: 10.1073/pnas.85.17.6277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis M. J., Pelham H. R. A human homologue of the yeast HDEL receptor. Nature. 1990 Nov 8;348(6297):162–163. doi: 10.1038/348162a0. [DOI] [PubMed] [Google Scholar]
- Lewis M. J., Pelham H. R. The sequence of the Kluyveromyces lactis BiP gene. Nucleic Acids Res. 1990 Nov 11;18(21):6438–6438. doi: 10.1093/nar/18.21.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis M. J., Sweet D. J., Pelham H. R. The ERD2 gene determines the specificity of the luminal ER protein retention system. Cell. 1990 Jun 29;61(7):1359–1363. doi: 10.1016/0092-8674(90)90699-f. [DOI] [PubMed] [Google Scholar]
- Machamer C. E., Doms R. W., Bole D. G., Helenius A., Rose J. K. Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein. J Biol Chem. 1990 Apr 25;265(12):6879–6883. [PubMed] [Google Scholar]
- Maundrell K., Nurse P., Schönholzer F., Schweingruber M. E. Cloning and characterization of two genes restoring acid phosphatase activity in pho1- mutants of Schizosaccharomyces pombe. Gene. 1985;39(2-3):223–230. doi: 10.1016/0378-1119(85)90316-6. [DOI] [PubMed] [Google Scholar]
- Miyake S., Yamamoto M. Identification of ras-related, YPT family genes in Schizosaccharomyces pombe. EMBO J. 1990 May;9(5):1417–1422. doi: 10.1002/j.1460-2075.1990.tb08257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
- Moreno S., Ruíz T., Sánchez Y., Villanueva J. R., Rodríguez L. Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe. Arch Microbiol. 1985 Sep;142(4):370–374. doi: 10.1007/BF00491906. [DOI] [PubMed] [Google Scholar]
- Munro S., Pelham H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987 Mar 13;48(5):899–907. doi: 10.1016/0092-8674(87)90086-9. [DOI] [PubMed] [Google Scholar]
- Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
- Munro S., Pelham H. R. Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. EMBO J. 1984 Dec 20;3(13):3087–3093. doi: 10.1002/j.1460-2075.1984.tb02263.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ng D. T., Randall R. E., Lamb R. A. Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface. J Cell Biol. 1989 Dec;109(6 Pt 2):3273–3289. doi: 10.1083/jcb.109.6.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Normington K., Kohno K., Kozutsumi Y., Gething M. J., Sambrook J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 1989 Jun 30;57(7):1223–1236. doi: 10.1016/0092-8674(89)90059-7. [DOI] [PubMed] [Google Scholar]
- Pelham H. R. Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J. 1988 Apr;7(4):913–918. doi: 10.1002/j.1460-2075.1988.tb02896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pelham H. R., Hardwick K. G., Lewis M. J. Sorting of soluble ER proteins in yeast. EMBO J. 1988 Jun;7(6):1757–1762. doi: 10.1002/j.1460-2075.1988.tb03005.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pelham H. R. The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci. 1990 Dec;15(12):483–486. doi: 10.1016/0968-0004(90)90303-s. [DOI] [PubMed] [Google Scholar]
- Resendez E., Jr, Wooden S. K., Lee A. S. Identification of highly conserved regulatory domains and protein-binding sites in the promoters of the rat and human genes encoding the stress-inducible 78-kilodalton glucose-regulated protein. Mol Cell Biol. 1988 Oct;8(10):4579–4584. doi: 10.1128/mcb.8.10.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
- Russell P., Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell. 1986 Apr 11;45(1):145–153. doi: 10.1016/0092-8674(86)90546-5. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Schweingruber A. M., Schoenholzer F., Keller L., Schwaninger R., Trachsel H., Schweingruber M. E. Glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe. Eur J Biochem. 1986 Jul 1;158(1):133–140. doi: 10.1111/j.1432-1033.1986.tb09730.x. [DOI] [PubMed] [Google Scholar]
- Schweingruber M. E., Fluri R., Maundrell K., Schweingruber A. M., Dumermuth E. Identification and characterization of thiamin repressible acid phosphatase in yeast. J Biol Chem. 1986 Dec 5;261(34):15877–15882. [PubMed] [Google Scholar]
- Semenza J. C., Hardwick K. G., Dean N., Pelham H. R. ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway. Cell. 1990 Jun 29;61(7):1349–1357. doi: 10.1016/0092-8674(90)90698-e. [DOI] [PubMed] [Google Scholar]
- Smith D. G., Svoboda A. Golgi apparatus in normal cells and protoplasts of Schizosaccharomyces pombe. Microbios. 1972 May-Jun;5(19):177–182. [PubMed] [Google Scholar]
- Tuite M. F., Bossier P., Fitch I. T. A highly conserved sequence in yeast heat shock gene promoters. Nucleic Acids Res. 1988 Dec 23;16(24):11845–11845. doi: 10.1093/nar/16.24.11845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaux D., Tooze J., Fuller S. Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal. Nature. 1990 Jun 7;345(6275):495–502. doi: 10.1038/345495a0. [DOI] [PubMed] [Google Scholar]
- Vogel J. P., Misra L. M., Rose M. D. Loss of BiP/GRP78 function blocks translocation of secretory proteins in yeast. J Cell Biol. 1990 Jun;110(6):1885–1895. doi: 10.1083/jcb.110.6.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watowich S. S., Morimoto R. I. Complex regulation of heat shock- and glucose-responsive genes in human cells. Mol Cell Biol. 1988 Jan;8(1):393–405. doi: 10.1128/mcb.8.1.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Welch W. J., Garrels J. I., Thomas G. P., Lin J. J., Feramisco J. R. Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins. J Biol Chem. 1983 Jun 10;258(11):7102–7111. [PubMed] [Google Scholar]
- Xiao H., Lis J. T. Germline transformation used to define key features of heat-shock response elements. Science. 1988 Mar 4;239(4844):1139–1142. doi: 10.1126/science.3125608. [DOI] [PubMed] [Google Scholar]
- Yanagida M. Yeast tubulin genes. Microbiol Sci. 1987 Apr;4(4):115–118. [PubMed] [Google Scholar]
- Yang J. W., Schweingruber M. E. The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe. Curr Genet. 1990 Oct;18(3):269–272. doi: 10.1007/BF00318392. [DOI] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]