Journal of Microbiology

Kwang-Lae Hoe

2 Articles

The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1: Dong-Uk Kim , Shinae Maeng , Hyemi Lee , Miyoung Nam , Sook-Jeong Lee , Kwang-Lae Hoe; J. Microbiol. 2016;54(2):98-105. Published online February 2, 2016; DOI: https://doi.org/10.1007/s12275-016-5569-y

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In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to β-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression.

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Generation of Expression Vectors for High-Throughput Functional Analysis of Target Genes in Schizosaccharomyces pombe: Jiwon Ahn , Chung-Hae Choi , Chang-Mo Kang , Chun-Ho Kim , Hee-Moon Park , Kyung-Bin Song , Kwang-Lae Hoe , Misun Won , Kyung-Sook Chung; J. Microbiol. 2009;47(6):789-795. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0010-4

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Abstract PDF

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, ura4+ and Saccharomyces cerevisiae LEU2 complementing leu1. These vectors contain 3 different strengths of the inducible promoter nmt1, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.

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