|
|
Searching for the Subcellular Targeting Sequences of Ste2,a GPCR Protein in Saccharomyces cerevisiae |
JIN Xue,SONG Jing-zhen,XIE Zhi-ping() |
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,Shanghai Jiao Tong University, Shanghai 200240, China |
|
|
Abstract Members of the G protein-coupled receptor (GPCR) family play important roles in the sensing of extracellular signals. Ste2 is one of the three GPCR proteins in the budding yeast, Saccharomyces cerevisiae. In the past years, extensive efforts have focused on how the function and expression of Ste2 are affected by various mutations. However, little is known about the mechanisms dictating its proper subcellular localization. To this end, a series of mutants containing deletions or substitutions in the N-terminus, C-terminus, transmembrane domains, intra/extracellular loops are constructed. The subcellular localization of wild-type and mutant Ste2 proteins are observed by fluorescent microscopy, in combination with a set of organelle markers, to determine their localization. Wild-type Ste2 is primarily targeted to the plasma membrane and vacuolar lumen. Deletion of the C-terminus eliminates the vascular signal, targeting the protein to the plasma membrane and endoplasmic reticulum instead. The result is similar when the C-terminus is substituted by the corresponding regions from ORI7 and OR17-40, two mammalian GPCRs. When the N-terminus, first extracellular loop (EL1), the second extracellular loop (EL2) or the third intracellular loop (IL3) is substituted, plasma membrane targeting of Ste2 is substantially attenuated or eliminated. Some of these mutants accumulate on intracellular punctate structures. These results suggest that the N-terminus, EL1, EL2 and IL3 regions contain potential sorting signals regulating the transport of Ste2 to the plasma membrane, and that the C-terminus contains signals for its targeting to the vacuole. The present work provides new insights towards understanding the mechanisms governing GPCR protein subcellular localization.
|
Received: 29 March 2019
Published: 17 December 2019
|
|
Corresponding Authors:
Zhi-ping XIE
E-mail: zxie@sjtu.edu.cn
|
|
|
[1] |
Fukutani Y, Ishii J, Kondo A , et al. Split luciferase complementation assay for the analysis of G protein‐coupled receptor ligand response in Saccharomyces cerevisiae. Biotechnology & Bioengineering, 2017,114(6):1354.
doi: 10.4014/jmb.1910.10055
pmid: 31838832
|
|
|
[2] |
Kristiansen K . Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function. Pharmacol Ther, 2004,103(1):21-80.
doi: 10.1016/j.pharmthera.2004.05.002
pmid: 15251227
|
|
|
[3] |
Stoneman M R, Paprocki J D, Biener G , et al. Quaternary structure of the yeast pheromone receptor Ste2 in living cells.Biochimica et Biophysica Acta (BBA) - Biomembranes, 2016,1859(9):1456-1464.
doi: 10.1016/j.bbamem.2016.12.008
pmid: 27993568
|
|
|
[4] |
Cevhero?lu O, Kumas G, Hauser M , et al. The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane.Biochimica et Biophysica Acta (BBA) - Biomembranes, 2017,1859(5):698-711.
doi: 10.1016/j.bbamem.2017.01.008
pmid: 28073700
|
|
|
[5] |
Naider F, Becker J M . The α-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors. Peptides, 2004,25(9):1441-1463.
doi: 10.1016/j.peptides.2003.11.028
|
|
|
[6] |
Bardwell L . A walk-through of the yeast mating pheromone response pathway. Peptides, 2004,25(9):1465-1476.
doi: 10.1016/j.peptides.2003.10.022
|
|
|
[7] |
Alvaro C G, Thorner J . Heterotrimeric G protein-coupled receptor signaling in yeast mating pheromone response. Journal of Biological Chemistry, 2016,291(15):7788-7795.
doi: 10.1074/jbc.R116.714980
pmid: 26907689
|
|
|
[8] |
Choudhary P, Loewen M C . Quantification of mutation-derived bias for alternate mating functionalities of the Saccharomyces cerevisiae Ste2p pheromone receptor. Journal of Biochemistry, 2016,159(1):49-58.
doi: 10.1093/jb/mvv072
pmid: 26232403
|
|
|
[9] |
Uddin M S, Naider F, Becker J M . Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2017,1859(10):2058-2067.
doi: 10.1016/j.bbamem.2017.07.014
pmid: 28754538
|
|
|
[10] |
Harley C A, Tipper D J . The Role of charged residues in determining transmembrane protein insertion orientation in yeast. Journal of Biological Chemistry, 1996,271(40):24625-24633.
doi: 10.1074/jbc.271.40.24625
pmid: 8798728
|
|
|
[11] |
Fukuda N, Kaishima M, Ishii J , et al. Positive detection of GPCR antagonists using a system for inverted expression of a fluorescent reporter gene. ACS Synthetic Biology, 2017,6(8):1554-1562.
doi: 10.1021/acssynbio.7b00056
pmid: 28499341
|
|
|
[12] |
Zecchin A, Stapor P C, Goveia J , et al. Metabolic pathway compartmentalization: an underappreciated opportunity. Curr Opin Biotechnol, 2015,34(34C):73-81.
doi: 10.1016/j.copbio.2014.11.022
pmid: 25499800
|
|
|
[13] |
Casad V, Corts A, Mallol J , et al. GPCR homomers and heteromers: A better choice as targets for drug development than GPCR monomers. Pharmacology & Therapeutics, 2009,124(2):248-257.
doi: 10.1007/s12035-019-01849-6
pmid: 31838720
|
|
|
[14] |
Rivero-Muller A, Chou Y Y, Ji I , et al. Rescue of defective G protein-coupled receptor function in vivo by intermolecular cooperation. Proceedings of the National Academy of Sciences, 2010,107(5):2319-2324.
|
|
|
[15] |
Uddin M S, Kim H, Deyo A , et al. Identification of residues involved in homodimer formation located within aβ-strand region of the N-terminus of a yeast G protein-coupled receptor. Journal of Receptor & Signal Transduction Research, 2012,32(2):65.
doi: 10.4014/jmb.1910.10055
pmid: 31838832
|
|
|
[16] |
Radhika V, Proikascezanne T, Jayaraman M , et al. Chemical sensing of DNT by engineered olfactory yeast strain. Nature Chemical Biology, 2007,3(6):325.
doi: 10.1038/nchembio882
pmid: 17486045
|
|
|
[17] |
Fukutani Y, Nakamura T, Yorozu M , et al. The N-terminal replacement of an olfactory receptor for the development of a yeast-based biomimetic odor sensor. Biotechnology and Bioengineering, 2012,109(1):205-212.
doi: 10.1002/bit.23327
|
|
|
[18] |
Minic J, Persuy M A, Godel E , et al. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS Journal, 2005,272(2):524-537.
doi: 10.1111/j.1742-4658.2004.04494.x
pmid: 15654890
|
|
|
[19] |
Seraj Uddin M, Hauser M, Naider F , et al. The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2016,1858(4):715-724.
doi: 10.1016/j.bbamem.2015.12.017
pmid: 26707753
|
|
|
[20] |
Kim K M, Lee Y H, Akal-Strader A , et al. Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR. Pharmacological Research, 2011,65(1):31-40.
doi: 10.1016/j.phrs.2011.11.002
pmid: 22100461
|
|
|
[21] |
Zuber J, Danial S A, Connelly S M , et al. Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae. Biochemistry, 2015,54(9):1787-1806.
doi: 10.1021/bi501314t
pmid: 25647246
|
|
|
[22] |
Gastaldi S, Zamboni M, Bolasco G , et al. Analysis of random PCR-originated mutants of the yeast Ste2 and Ste3 receptors. Microbiologyopen, 2016,5(4):670-686.
doi: 10.1002/mbo3.361
pmid: 27150158
|
|
|
[23] |
Sridharan R, Connelly S M, Naider F , et al. Variable dependence of signaling output on agonist occupancy of Ste2p, a G protein coupled receptor in yeast. Journal of Biological Chemistry, 2016,29(46):24261-24279.
doi: 10.1074/jbc.M116.733006
pmid: 27646004
|
|
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|