Single-amino-acid Substitutions Within the Signal Sequence of Yeast Prepro-alpha-factor Affect Membrane Translocation

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1988 May 1

PMID 3290645

Citations 10

Authors

D S Allison

E T Young

Affiliations

Soon will be listed here.

Abstract

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.

Citing Articles

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

Robeson L, Casanova-Morales N, Burgos-Bravo F, Alfaro-Valdes H, Lesch R, Ramirez-Alvarez C Protein Sci. 2024; 33(6):e4996.

PMID: 38747383 PMC: 11094780. DOI: 10.1002/pro.4996.

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae.

Cruz-Teran C, Tiruthani K, Mischler A, Rao B ACS Synth Biol. 2017; 6(11):2096-2107.

PMID: 28805373 PMC: 5905331. DOI: 10.1021/acssynbio.7b00144.

Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum.

Dunnwald M, Varshavsky A, Johnsson N Mol Biol Cell. 1999; 10(2):329-44.

PMID: 9950680 PMC: 25172. DOI: 10.1091/mbc.10.2.329.

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation.

Ngsee J, Hansen W, Walter P, Smith M Mol Cell Biol. 1989; 9(8):3400-10.

PMID: 2677671 PMC: 362386. DOI: 10.1128/mcb.9.8.3400-3410.1989.

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

Allison D, Young E Mol Cell Biol. 1989; 9(11):4977-85.

PMID: 2513481 PMC: 363649. DOI: 10.1128/mcb.9.11.4977-4985.1989.

References

Emr S, Silhavy T . Importance of secondary structure in the signal sequence for protein secretion. Proc Natl Acad Sci U S A. 1983; 80(15):4599-603. PMC: 384091. DOI: 10.1073/pnas.80.15.4599. View

Silve S, Monod M, Hinnen A, Haguenauer-Tsapis R . The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence. Mol Cell Biol. 1987; 7(9):3306-14. PMC: 367968. DOI: 10.1128/mcb.7.9.3306-3314.1987. View

Suissa M . Spectrophotometric quantitation of silver grains eluted from autoradiograms. Anal Biochem. 1983; 133(2):511-4. DOI: 10.1016/0003-2697(83)90117-3. View

Shaw K, Olson M . Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene. Mol Cell Biol. 1984; 4(4):657-65. PMC: 368776. DOI: 10.1128/mcb.4.4.657-665.1984. View

Maarse A, van Loon A, Riezman H, Gregor I, Schatz G, Grivell L . Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein. EMBO J. 1984; 3(12):2831-7. PMC: 557773. DOI: 10.1002/j.1460-2075.1984.tb02216.x. View

Hurt E, Schatz G . The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984; 3(13):3149-56. PMC: 557831. DOI: 10.1002/j.1460-2075.1984.tb02272.x. View

Brown P, HALVORSON H, Raney P, PERLMAN D . Conformational alterations in the proximal portion of the yeast invertase signal peptide do not block secretion. Mol Gen Genet. 1984; 197(3):351-7. DOI: 10.1007/BF00329928. View

Schauer I, Emr S, Gross C, Schekman R . Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985; 100(5):1664-75. PMC: 2113855. DOI: 10.1083/jcb.100.5.1664. View

Kurjan J . Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. Mol Cell Biol. 1985; 5(4):787-96. PMC: 366783. DOI: 10.1128/mcb.5.4.787-796.1985. View

10.

Briggs M, Gierasch L, Zlotnick A, Lear J, Degrado W . In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides. Science. 1985; 228(4703):1096-9. DOI: 10.1126/science.3158076. View

11.

Myers R, LERMAN L, Maniatis T . A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985; 229(4710):242-7. DOI: 10.1126/science.2990046. View

12.

Dowhan W, Bibus C, Schatz G . The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast. EMBO J. 1985; 4(1):179-84. PMC: 554168. DOI: 10.1002/j.1460-2075.1985.tb02334.x. View

13.

Benson S, Hall M, Silhavy T . Genetic analysis of protein export in Escherichia coli K12. Annu Rev Biochem. 1985; 54:101-34. DOI: 10.1146/annurev.bi.54.070185.000533. View

14.

von Heijne G . Signal sequences. The limits of variation. J Mol Biol. 1985; 184(1):99-105. DOI: 10.1016/0022-2836(85)90046-4. View

15.

Hurt E, Suda K, Oppliger W, Schatz G . The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 1985; 4(8):2061-8. PMC: 554462. DOI: 10.1002/j.1460-2075.1985.tb03892.x. View

16.

Rothblatt J, Meyer D . Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986; 44(4):619-28. DOI: 10.1016/0092-8674(86)90271-0. View

17.

van Loon A, Young E . Intracellular sorting of alcohol dehydrogenase isoenzymes in yeast: a cytosolic location reflects absence of an amino-terminal targeting sequence for the mitochondrion. EMBO J. 1986; 5(1):161-5. PMC: 1166709. DOI: 10.1002/j.1460-2075.1986.tb04191.x. View

18.

Hansen W, Garcia P, Walter P . In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986; 45(3):397-406. DOI: 10.1016/0092-8674(86)90325-9. View

19.

Waters M, Blobel G . Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986; 102(5):1543-50. PMC: 2114218. DOI: 10.1083/jcb.102.5.1543. View

20.

PERLMAN D, Raney P, HALVORSON H . Mutations affecting the signal sequence alter synthesis and secretion of yeast invertase. Proc Natl Acad Sci U S A. 1986; 83(14):5033-7. PMC: 323884. DOI: 10.1073/pnas.83.14.5033. View