Abstract
The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.
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- Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ammerer G. Sex, stress and integrity: the importance of MAP kinases in yeast. Curr Opin Genet Dev. 1994 Feb;4(1):90–95. doi: 10.1016/0959-437x(94)90096-5. [DOI] [PubMed] [Google Scholar]
- Bardwell L., Cook J. G., Inouye C. J., Thorner J. Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev Biol. 1994 Dec;166(2):363–379. doi: 10.1006/dbio.1994.1323. [DOI] [PubMed] [Google Scholar]
- Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell. 1992 Dec 24;71(7):1069–1072. doi: 10.1016/s0092-8674(05)80056-x. [DOI] [PubMed] [Google Scholar]
- Blumer K. J., Reneke J. E., Thorner J. The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. J Biol Chem. 1988 Aug 5;263(22):10836–10842. [PubMed] [Google Scholar]
- Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
- Burkholder A. C., Hartwell L. H. The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res. 1985 Dec 9;13(23):8463–8475. doi: 10.1093/nar/13.23.8463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang F., Herskowitz I. Phosphorylation of FAR1 in response to alpha-factor: a possible requirement for cell-cycle arrest. Mol Biol Cell. 1992 Apr;3(4):445–450. doi: 10.1091/mbc.3.4.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chant J. Cell polarity in yeast. Trends Genet. 1994 Sep;10(9):328–333. doi: 10.1016/0168-9525(94)90036-1. [DOI] [PubMed] [Google Scholar]
- Chenevert J. Cell polarization directed by extracellular cues in yeast. Mol Biol Cell. 1994 Nov;5(11):1169–1175. doi: 10.1091/mbc.5.11.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chenevert J., Corrado K., Bender A., Pringle J., Herskowitz I. A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains. Nature. 1992 Mar 5;356(6364):77–79. doi: 10.1038/356077a0. [DOI] [PubMed] [Google Scholar]
- Chenevert J., Valtz N., Herskowitz I. Identification of genes required for normal pheromone-induced cell polarization in Saccharomyces cerevisiae. Genetics. 1994 Apr;136(4):1287–1296. doi: 10.1093/genetics/136.4.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choi K. Y., Satterberg B., Lyons D. M., Elion E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994 Aug 12;78(3):499–512. doi: 10.1016/0092-8674(94)90427-8. [DOI] [PubMed] [Google Scholar]
- Clark K. L., Dignard D., Thomas D. Y., Whiteway M. Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating. Mol Cell Biol. 1993 Jan;13(1):1–8. doi: 10.1128/mcb.13.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Courchesne W. E., Kunisawa R., Thorner J. A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae. Cell. 1989 Sep 22;58(6):1107–1119. doi: 10.1016/0092-8674(89)90509-6. [DOI] [PubMed] [Google Scholar]
- Dietzel C., Kurjan J. The yeast SCG1 gene: a G alpha-like protein implicated in the a- and alpha-factor response pathway. Cell. 1987 Sep 25;50(7):1001–1010. doi: 10.1016/0092-8674(87)90166-8. [DOI] [PubMed] [Google Scholar]
- Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
- Dolan J. W., Fields S. Overproduction of the yeast STE12 protein leads to constitutive transcriptional induction. Genes Dev. 1990 Apr;4(4):492–502. doi: 10.1101/gad.4.4.492. [DOI] [PubMed] [Google Scholar]
- Dolan J. W., Kirkman C., Fields S. The yeast STE12 protein binds to the DNA sequence mediating pheromone induction. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5703–5707. doi: 10.1073/pnas.86.15.5703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dorer R., Pryciak P. M., Hartwell L. H. Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients. J Cell Biol. 1995 Nov;131(4):845–861. doi: 10.1083/jcb.131.4.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drubin D. G., Nelson W. J. Origins of cell polarity. Cell. 1996 Feb 9;84(3):335–344. doi: 10.1016/s0092-8674(00)81278-7. [DOI] [PubMed] [Google Scholar]
- Elion E. A., Brill J. A., Fink G. R. FUS3 represses CLN1 and CLN2 and in concert with KSS1 promotes signal transduction. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9392–9396. doi: 10.1073/pnas.88.21.9392. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elion E. A., Grisafi P. L., Fink G. R. FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation. Cell. 1990 Feb 23;60(4):649–664. doi: 10.1016/0092-8674(90)90668-5. [DOI] [PubMed] [Google Scholar]
- Errede B., Ammerer G. STE12, a protein involved in cell-type-specific transcription and signal transduction in yeast, is part of protein-DNA complexes. Genes Dev. 1989 Sep;3(9):1349–1361. doi: 10.1101/gad.3.9.1349. [DOI] [PubMed] [Google Scholar]
- Errede B., Gartner A., Zhou Z., Nasmyth K., Ammerer G. MAP kinase-related FUS3 from S. cerevisiae is activated by STE7 in vitro. Nature. 1993 Mar 18;362(6417):261–264. doi: 10.1038/362261a0. [DOI] [PubMed] [Google Scholar]
- Ferguson B., Horecka J., Printen J., Schultz J., Stevenson B. J., Sprague G. F., Jr The yeast pheromone response pathway: new insights into signal transmission. Cell Mol Biol Res. 1994;40(3):223–228. [PubMed] [Google Scholar]
- Field C., Schekman R. Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J Cell Biol. 1980 Jul;86(1):123–128. doi: 10.1083/jcb.86.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hagen D. C., McCaffrey G., Sprague G. F., Jr Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1418–1422. doi: 10.1073/pnas.83.5.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartwell L. H. Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J Cell Biol. 1980 Jun;85(3):811–822. doi: 10.1083/jcb.85.3.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hasson M. S., Blinder D., Thorner J., Jenness D. D. Mutational activation of the STE5 gene product bypasses the requirement for G protein beta and gamma subunits in the yeast pheromone response pathway. Mol Cell Biol. 1994 Feb;14(2):1054–1065. doi: 10.1128/mcb.14.2.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herlitze S., Garcia D. E., Mackie K., Hille B., Scheuer T., Catterall W. A. Modulation of Ca2+ channels by G-protein beta gamma subunits. Nature. 1996 Mar 21;380(6571):258–262. doi: 10.1038/380258a0. [DOI] [PubMed] [Google Scholar]
- Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell. 1995 Jan 27;80(2):187–197. doi: 10.1016/0092-8674(95)90402-6. [DOI] [PubMed] [Google Scholar]
- Iida H., Nakamura H., Ono T., Okumura M. S., Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Mol Cell Biol. 1994 Dec;14(12):8259–8271. doi: 10.1128/mcb.14.12.8259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iida H., Yagawa Y., Anraku Y. Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2. J Biol Chem. 1990 Aug 5;265(22):13391–13399. [PubMed] [Google Scholar]
- Jackson C. L., Hartwell L. H. Courtship in S. cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal. Cell. 1990 Nov 30;63(5):1039–1051. doi: 10.1016/0092-8674(90)90507-b. [DOI] [PubMed] [Google Scholar]
- Jackson C. L., Hartwell L. H. Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating. Mol Cell Biol. 1990 May;10(5):2202–2213. doi: 10.1128/mcb.10.5.2202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson C. L., Konopka J. B., Hartwell L. H. S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination. Cell. 1991 Oct 18;67(2):389–402. doi: 10.1016/0092-8674(91)90190-a. [DOI] [PubMed] [Google Scholar]
- Jenness D. D., Burkholder A. C., Hartwell L. H. Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites. Mol Cell Biol. 1986 Jan;6(1):318–320. doi: 10.1128/mcb.6.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kao L. R., Peterson J., Ji R., Bender L., Bender A. Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jan;16(1):168–178. doi: 10.1128/mcb.16.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Konopka J. B., Jenness D. D., Hartwell L. H. The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone. Cell. 1988 Aug 26;54(5):609–620. doi: 10.1016/s0092-8674(88)80005-9. [DOI] [PubMed] [Google Scholar]
- Kranz J. E., Satterberg B., Elion E. A. The MAP kinase Fus3 associates with and phosphorylates the upstream signaling component Ste5. Genes Dev. 1994 Feb 1;8(3):313–327. doi: 10.1101/gad.8.3.313. [DOI] [PubMed] [Google Scholar]
- Kurjan J. Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. Mol Cell Biol. 1985 Apr;5(4):787–796. doi: 10.1128/mcb.5.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kurjan J. Pheromone response in yeast. Annu Rev Biochem. 1992;61:1097–1129. doi: 10.1146/annurev.bi.61.070192.005313. [DOI] [PubMed] [Google Scholar]
- Kurjan J. The pheromone response pathway in Saccharomyces cerevisiae. Annu Rev Genet. 1993;27:147–179. doi: 10.1146/annurev.ge.27.120193.001051. [DOI] [PubMed] [Google Scholar]
- Leberer E., Dignard D., Hougan L., Thomas D. Y., Whiteway M. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling. EMBO J. 1992 Dec;11(13):4805–4813. doi: 10.1002/j.1460-2075.1992.tb05586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leeuw T., Fourest-Lieuvin A., Wu C., Chenevert J., Clark K., Whiteway M., Thomas D. Y., Leberer E. Pheromone response in yeast: association of Bem1p with proteins of the MAP kinase cascade and actin. Science. 1995 Nov 17;270(5239):1210–1213. doi: 10.1126/science.270.5239.1210. [DOI] [PubMed] [Google Scholar]
- Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
- Lyons D. M., Mahanty S. K., Choi K. Y., Manandhar M., Elion E. A. The SH3-domain protein Bem1 coordinates mitogen-activated protein kinase cascade activation with cell cycle control in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4095–4106. doi: 10.1128/mcb.16.8.4095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Madden K., Snyder M. Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection. Mol Biol Cell. 1992 Sep;3(9):1025–1035. doi: 10.1091/mbc.3.9.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marcus S., Polverino A., Barr M., Wigler M. Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7762–7766. doi: 10.1073/pnas.91.16.7762. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michaelis S., Herskowitz I. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol. 1988 Mar;8(3):1309–1318. doi: 10.1128/mcb.8.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyajima I., Nakafuku M., Nakayama N., Brenner C., Miyajima A., Kaibuchi K., Arai K., Kaziro Y., Matsumoto K. GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell. 1987 Sep 25;50(7):1011–1019. doi: 10.1016/0092-8674(87)90167-x. [DOI] [PubMed] [Google Scholar]
- Nakayama N., Miyajima A., Arai K. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 1985 Oct;4(10):2643–2648. doi: 10.1002/j.1460-2075.1985.tb03982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ono T., Suzuki T., Anraku Y., Iida H. The MID2 gene encodes a putative integral membrane protein with a Ca(2+)-binding domain and shows mating pheromone-stimulated expression in Saccharomyces cerevisiae. Gene. 1994 Dec 30;151(1-2):203–208. doi: 10.1016/0378-1119(94)90657-2. [DOI] [PubMed] [Google Scholar]
- Peter M., Gartner A., Horecka J., Ammerer G., Herskowitz I. FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell. 1993 May 21;73(4):747–760. doi: 10.1016/0092-8674(93)90254-n. [DOI] [PubMed] [Google Scholar]
- Peterson J., Zheng Y., Bender L., Myers A., Cerione R., Bender A. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J Cell Biol. 1994 Dec;127(5):1395–1406. doi: 10.1083/jcb.127.5.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pringle J. R., Bi E., Harkins H. A., Zahner J. E., De Virgilio C., Chant J., Corrado K., Fares H. Establishment of cell polarity in yeast. Cold Spring Harb Symp Quant Biol. 1995;60:729–744. doi: 10.1101/sqb.1995.060.01.079. [DOI] [PubMed] [Google Scholar]
- Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
- Printen J. A., Sprague G. F., Jr Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics. 1994 Nov;138(3):609–619. doi: 10.1093/genetics/138.3.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramer S. W., Davis R. W. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):452–456. doi: 10.1073/pnas.90.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reid B. J., Hartwell L. H. Regulation of mating in the cell cycle of Saccharomyces cerevisiae. J Cell Biol. 1977 Nov;75(2 Pt 1):355–365. doi: 10.1083/jcb.75.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhodes N., Connell L., Errede B. STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast. Genes Dev. 1990 Nov;4(11):1862–1874. doi: 10.1101/gad.4.11.1862. [DOI] [PubMed] [Google Scholar]
- Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
- Segall J. E. Polarization of yeast cells in spatial gradients of alpha mating factor. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8332–8336. doi: 10.1073/pnas.90.18.8332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon M. N., De Virgilio C., Souza B., Pringle J. R., Abo A., Reed S. I. Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway. Nature. 1995 Aug 24;376(6542):702–705. doi: 10.1038/376702a0. [DOI] [PubMed] [Google Scholar]
- Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sloat B. F., Pringle J. R. A mutant of yeast defective in cellular morphogenesis. Science. 1978 Jun 9;200(4346):1171–1173. doi: 10.1126/science.349694. [DOI] [PubMed] [Google Scholar]
- Teague M. A., Chaleff D. T., Errede B. Nucleotide sequence of the yeast regulatory gene STE7 predicts a protein homologous to protein kinases. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7371–7375. doi: 10.1073/pnas.83.19.7371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thorburn J., Frost J. A., Thorburn A. Mitogen-activated protein kinases mediate changes in gene expression, but not cytoskeletal organization associated with cardiac muscle cell hypertrophy. J Cell Biol. 1994 Sep;126(6):1565–1572. doi: 10.1083/jcb.126.6.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trueheart J., Boeke J. D., Fink G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol. 1987 Jul;7(7):2316–2328. doi: 10.1128/mcb.7.7.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyers M., Futcher B. Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes. Mol Cell Biol. 1993 Sep;13(9):5659–5669. doi: 10.1128/mcb.13.9.5659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valdivieso M. H., Sugimoto K., Jahng K. Y., Fernandes P. M., Wittenberg C. FAR1 is required for posttranscriptional regulation of CLN2 gene expression in response to mating pheromone. Mol Cell Biol. 1993 Feb;13(2):1013–1022. doi: 10.1128/mcb.13.2.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valtz N., Peter M., Herskowitz I. FAR1 is required for oriented polarization of yeast cells in response to mating pheromones. J Cell Biol. 1995 Nov;131(4):863–873. doi: 10.1083/jcb.131.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
- Wu C., Whiteway M., Thomas D. Y., Leberer E. Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. J Biol Chem. 1995 Jul 7;270(27):15984–15992. doi: 10.1074/jbc.270.27.15984. [DOI] [PubMed] [Google Scholar]