Characterization of yeast Yea4p, a uridine diphosphate-N-acetylglucosamine transporter localized in the endoplasmic reticulum and required for chitin synthesis

Chitin is an essential cell wall component, synthesis of which is regulated throughout the cell cycle in the yeast Saccharomyces cerevisiae. We cloned an S, cerevisiae gene, YEA4, whose product is homologous to the Kluyueromyces lactis uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transporter. An epitope-tagged Yea4p localized mainly in the 10,000 x g pellet (P2), suggesting endoplasmic reticulum (ER) localization. Membrane vesicles from the P2 fraction showed an 8-fold higher UDP-GlcNAc transport activity in cells harboring a multicopy YEA4 plasmid than in cells harboring vector alone, The activity distribution is identical with the protein distribution in P2, whether the gene is overexpressed or not, suggesting its native localization in P2. Immunolocalization of epitope-tagged Yea4p further revealed ER localization. The increase in transport activity due to the YEA4 overexpression is specific for UDP-GlcNAc, but not for UDP-galactose and GDP-mannose, Delta yea4-disrupted cells showed a reduced rate of UDP-GlcNAc transport, contained less chitin, and were larger and rounder in shape than the wild type cells. Our results indicate that YEA4 encodes an ER-localized UDP-GlcNAc transporter that is required for cell wall chitin synthesis in S. cerevisiae.