This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poulsen, P.
Right arrow Articles by Kielland-Brandt, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poulsen, P.
Right arrow Articles by Kielland-Brandt, M. C.

 Previous Article  |  Next Article 

Eukaryotic Cell, March 2006, p. 601-608, Vol. 5, No. 3
1535-9778/06/$08.00+0     doi:10.1128/EC.5.3.601-608.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of Saccharomyces cerevisiae and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics

Peter Poulsen,1 Leila Lo Leggio,2 and Morten C. Kielland-Brandt1*

Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark,1 Centre for Crystallographic Studies, Biophysical Chemistry Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark2

Received 28 November 2005/ Accepted 11 January 2006

Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC50) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC50 of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with {alpha}-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.

* Corresponding author. Mailing address: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark. Phone: (45) 3327 5331. Fax: (45) 3327 4765. E-mail: mkb{at}crc.dk.

Eukaryotic Cell, March 2006, p. 601-608, Vol. 5, No. 3
1535-9778/06/$08.00+0     doi:10.1128/EC.5.3.601-608.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hundal, H. S., Taylor, P. M. (2009). Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling. Am. J. Physiol. Endocrinol. Metab. 296: E603-E613 [Abstract] [Full Text]  
  • Hwang, C.-S., Varshavsky, A. (2008). Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway. Proc. Natl. Acad. Sci. USA 105: 19188-19193 [Abstract] [Full Text]  
  • Liu, Z., Thornton, J., Spirek, M., Butow, R. A. (2008). Activation of the SPS Amino Acid-Sensing Pathway in Saccharomyces cerevisiae Correlates with the Phosphorylation State of a Sensor Component, Ptr3. Mol. Cell. Biol. 28: 551-563 [Abstract] [Full Text]  
  • Andreasson, C., Heessen, S., Ljungdahl, P. O. (2006). Regulation of transcription factor latency by receptor-activated proteolysis.. Genes Dev. 20: 1563-1568 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»