Characterization of Yeast Yea4p, a Uridine Diphosphate-N-acetylglucosamine Transporter Localized in the Endoplasmic Reticulum and Required for Chitin Synthesis

Abstract

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 Kluyveromyces lactis uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transporter. An epitope-tagged Yea4p localized mainly in the 10,000 ؋ 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 GDPmannose. ⌬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 ERlocalized UDP-GlcNAc transporter that is required for cell wall chitin synthesis in S. cerevisiae.The cell wall of Saccharomyces cerevisiae is composed of three types of polysaccharides: mannoprotein, ␤-glucan, and chitin. Chitin, a linear homopolymer of ␤-1,4-linked N-acetylglucosamine (GlcNAc) residues, is a relatively minor component, representing only 1-2% of the dry weight of the wall of vegetative cells (1). The biosynthesis of mannoprotein polysaccharides is well established; it initiates in the ER 1 and continues in the Golgi with the transfer of additional mannose units (2). ␤-Glucan synthesis is believed to take place along the secretory pathway in the ER and Golgi compartments and in the plasma membrane (3-5). In contrast, chitin synthesis is a complex process and not yet well understood (6 -8), although the enzymatic synthesis of chitin has been studied extensively (9). Chitin synthesis requires the activities of several different chitin synthases and many different genes for the control of these enzymes (10).S. cerevisiae possesses three chitin synthases. Chitin synthases I and II (CSI and CSII), the products of the CHS1 and CHS2, are closely related proteins and require partial proteolysis for their activity in vitro. In contrast, chitin synthase III (CSIII), the product of CHS3 (recently renamed from CAL1/ CSD2/DIT101/KTI2; Ref. 3), is active without proteolytic treatment and is presumed to be the catalytic subunit of a complex containing the CHS4 (CAL2/CSD4) and CHS5 (CAL3) gene products, which are believed to play regulatory roles (10 -12). The three chitin synthases have unique functions. CSI and CSII make only a small portion (Ͻ10%) of the total chitin, whereas CSIII is responsible for the remainder (Ͼ90%). Hydropathy analysis of the Chs1p, Chs2p, and Chs3p sequences reveals the presence of putative multimembrane-spanning domains in each polyp...

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein‐folding process at the endoplasmic reticulum

“…Further, scHut1p and spHut1p have a common sequence motif for the ER retention at their C-termini ( Figure 1A). S. cerevisiae UDP-N-acetylglucosamine transporter, Yea4p, has this motif (KKXX) at the C-terminus and is confirmed to be localized in the ER (Roy et al, 2000). While the C-terminal sequence of scHut1p (KAKSA) dose not completely fit to KKXX motif, KXKXX sequence can also work as an ER retention signal (Jackson et.…”

“…The pART1 plasmid (McLeod et al, 1987) was used to express the scHUT1-3rHA fusion gene, which contains the 3rHA epitope tag sequence at the 3k-end of the scHUT1 ORF in S. pombe. The oligonucleotide primers, 5k-CCGGAGCTCAAAAAAATGGCGGG AAGTACATCC-3k and 5k-AAACATGCGGCC GCCCGCAGATTTTGCCTT-3k, were used to amplify the scHUT1 ORF and the PCR product was cloned into SacI-and NotI-digested pYEX-3HA (Roy et al, 2000). The SacI and SphI fragment containing scHUT1-3HA fusion was ligated into SacI-and SphI-digested pSP73 (Promega).…”

“…The samples were subjected to SDS-PAGE. Western blot analysis was carried out using anti-scHut1p antibody, antiAnp1p antibody (a generous gift from Sean Munro) and anti-Dpm1p antibody (Molecular Probes), as described previously (Roy et al, 2000). Anti-scHut1p antibody was raised against a synthetic oligopeptide (CWEALNKKKANIPKAKSA) which corresponds to the C-terminal sequence of scHut1p.…”

Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

“…4 and S1). Moreover, the C-terminal domain of several NSTs has been implicated in targeting NSTs to the ER and/or Golgi apparatus, through dilysine retention and/or hydrophobic export motifs (66,69,70). Both LPG5A and LPG5B show potential motifs of this sort (Fig.…”

Molecular Cloning and Identification of 3′-Phosphoadenosine 5′-Phosphosulfate Transporter

“…Transformed yeast cells were grown at 30°C in synthetic defined medium in which uracil was omitted for the selection of transformants. Subcellular fractionation and nucleotide-sugar transport assays were performed as described by Roy et al (19). Yeast cells were converted into spheroplasts, homogenized, and fractionated to yield a 10,000 ϫ g membrane fraction (P10), a 100,000 ϫ g membrane fraction (P100), and the supernatant of the cytosolic fraction (S100).…”