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NISR RESEARCH GRANT
2004 Research Grant

The Process of Cell Wall Synthesis That Depends on Vesicular Transport
Koji YODA
Graduate School of Agricultural and Life Sciences, The University of Tokyo
Research aims

Results

The cells of microorganisms have a rigid cell wall attheir surface and thus, are able to live in various harsh environments. The fungi and yeast are eukaryotic microorganisms and have a cell wall composed of complex polysaccharides and proteins. Chitin and b -1,3-glucan are synthesized by enzymes in the cell membrane directly from theirsoluble precursors. On the contrary, b -1,6-glucan andmannoproteins are subjected to maturation and proper localization processes after their synthesis via the intracellular vesicular transport. This starts from the endoplasmic
reticulum (ER), through the Golgi apparatus and to the cellsurface. These processes are yet not fully clear. In thisstudy, we discovered a number of novel proteins from theisolated organelles or mutants with a defective cell wall,and characterized them. These results will provide muchunderstandings as to the metabolism, growth, cellular functions and biotechnological application of fungi and yeast.

Rcr1 protein The gene product of RCR1 which induces
resistance to Congo red by its multicopy introduction, wasan integral membrane protein in the ER. It actually works
in the wild-type cell because its null mutant showed an increased sensitivity to Congo red. By the introduction ofmulticopy RCR1 in various yeast mutants with various
Congo red sensitivity, the chitin content in the cell wall significantly decreased while the glucan content did notchange. No alteration was found in the amount and localization of chitin synthases. The ER membrane protein Rcr1
regulates the chitin synthases at the cell membrane bymechanisms still unknown. It was confirmed that the major
target of Congo red in yeast is also chitin and not glucan(Imai et al., 2005).
Skg1 and Skg6 proteins Novel proteins Skg1 andSkg6 were discovered by our multicopy suppressor screening of the synthetic lethality of the of kex2D gas1D mutations. They are integral membrane proteins and localize inthe growing region of the budding cell. These proteins arelikely to help the polarized growth of the cell wall
(Tomishige et al., 2005a, 2005b).
Svp26 protein Although the yeast Golgi apparatus iscomposed of multiple subcompartments with different protein composition and function, the exact analysis of theirprotein composition has not been done before. We succeeded in the isolation and purification of the early and lateGolgi compartments by immunoadsorption. The isolated
vesicles had different transport-related proteins, glycosylation enzymes, and glucan-synthetic proteins. We discovered
six novel membrane proteins. The Svp26 protein in theearly Golgi compartment was an integral membrane protein. It bound to Svp26 itself and to mannosyltransferase
Ktr3. In the svp26D null mutant, we found a shift in the localization of various Golgi membrane proteins. The majority of Ktr3 was found in the ER in the svp26D cell. Furthermore, the degree of outer N-glycosylation of secretory proteins significantly increased in the cell. Therefore, Svp26
regulates the localization of Golgi proteins and the processof N-glycan elongation (Inadome et al., 2005).

Methods
The budding yeast Saccharomyces cerevisiae was routinely used in this study.
A novel gene RCR1 was found by induction of resistanceto an antifungal dye Congo red which was believed to bindto the yeast cell wall glucan and inhibit its assembly. A simultaneous loss of Gas1 and Kex2 which plays a role in thematuration of glucan and processing of secretory proteins,respectively, is synthetically lethal. The novel genes SKG1and SKG6 were isolated among the multicopy suppressor
genes of this phenotype. The gene products were taggedwith epitope, their intracellular localization was observedby indirect immunofluorescent staining, and the phenotypesof their disruptants, chimera mutants or multicopy carriers,were examined.
The intracellular vesicles of the yeast Golgi apparatus
were immunoisolated using tagged membrane proteins
Sed5 or Tlg2 as the marker after mild cell disintegration.
Thus the early and late Golgi compartments were successfully isolated. The protein constituents were identified by
micropeptide mapping using MALDI TOF-MS. Six novel
proteins were characterized by epitope tagging and genedisruption to find out their interactions and roles in vesicular trafficking.

Conclusion
Our genetic and biochemical study of yeast discovered
novel membrane proteins in the ER and Golgi, and wemade important findings regarding their roles in the cell85

wall chitin content, structure of glycoproteins and polarizedcell growth.
References
Imai, K., N., Noda, Y., Adachi, H. and Yoda, K. (2005) A novel ER membrane protein Rcr1 regulates chitin deposition in the cell wall of Saccharomyces cerevisiae. J. Biol. Chem., 280, 8275–8284.
Tomishige, N., Noda, Y., Adachi, H., Shimoi, H. and Yoda, K. (2005a)
SKG1, a suppressor gene of synthetic lethality of kex2D gas1D muta-tions, encodes a novel membrane protein that affects cell wall composition. Yeast, 22, 141–155.
Tomishige, N., Noda, Y., Adachi, H. and Yoda, K. (2005b) SKG6, a suppressor gene of synthetic lethality of kex2D gas1D mutations, encodes anovel membrane protein showing polarized intracellular localization. J.
Gen. Appl. Microbiol., 51, in press.
Inadome, H., Noda, Y., Adachi, H. and Yoda, K. (2005) Immunoisolationof the yeast Golgi subcompartments and characterization of a novelmembrane protein, Svp26, discovered in the Sed5-containing compartments. Mol. Cell. Biol., 25, 7696–7710.