The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae
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The integrity of the Saccharomyces cerevisiae cell wall requires a functional Pkc1–Slt2 MAP kinase pathway that contributes to transient growth arrest, enabling coordination of cell division with cell wall remodelling. How this coordination takes place is still an open question. Recently, we brought evidence that Knr4 protein, whose absence leads to several cell wall defects, may play a role in this function. Here, we show that Knr4 is a monomeric protein that exhibits an aberrant mobility on a SDS-gel electrophoresis and a non-globular structure. Furthermore, Knr4 is an unstable protein that is degraded as cells enter the stationary phase of growth, while its corresponding gene is constitutively expressed. In exponentially growing cells on glucose, Knr4 appeared to be present in a protein complex that migrates with an apparent Mw superior to 250 kDa. Using the TAP–tag methodology, nine potential partners of Knr4 were identified, which could be distributed into three biological processes. A first group consisted of Slt2 and Pil1, two proteins dedicated to cell wall maintenance and biogenesis. The second group comprised four proteins (Bud6, Act1, Cin8 and Jnm1) implicated in the establishment of cell polarity and bud integrity during mitosis. The last group contained four proteins (Asc1, Ubc1, Hsc82 and Gvp36) that probably deal with the stability/degradation of proteins. Deletion analysis revealed that the domain of interaction covered 2/3 of the Knr4 sequence on the N-terminal side. Moreover, the replacement of the two in vivo phosphorylated Ser200 and Ser203 by alanines led to a mutated protein with reduced protein interactions and a weaker complementation ability towards knr4 null mutant phenotypes. These results together with previous data from genome scale two-hybrid and synthetic interaction screens support the notion that Knr4 is a regulatory protein that participates in the coordination of cell wall synthesis with bud emergence, and that this function may be modulated by phosphorylation of this protein.
KeywordsKNR4/SMI1 Protein interaction TAP–tag Two-hybrid Cell wall Signalization
We thank our colleagues and in particular Dr. Jean Luc Parrou for his help with RT-PCR experiments. We are grateful to M. Crouzet (LBMS, Bordeaux, France), J. Heinisch (University of Osnabrück, Germany ), K. Matsumoto (Nagoya University, Japan) and M. Molina (CSIC, Facultad de Farmacia, Madrid) for kind provision of yeast strains and plasmids. We also thank Miss Faubladier for helping us with the sucrose gradient experiment in Fig. 2. This work was supported in part by grants (QLK3-CT2000-01537) from the European Commission Framework Program and from ‘Fonds de Recherche Hoechst Marion Roussel’ (FRHMR2/9922) to JF. F.B. holds a pre-doctoral grant from Syrian Ministry of Research and Education.
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