The NAG1 gene is nested opposite the YGR031W ORF and encodes a protein product. (A) Schematic of the transposon-based gene trap used to identify YGR031C-A/NAG1. The lacZ reporter lacks its start codon and promoter, so β-galactosidase is produced only if the transposon inserts in genomic DNA such that the lacZ coding sequence is in frame with a host gene. The transposon-encoded lacZ fusion will be separated from host gene coding sequence by the Tn3L terminal sequence and loxR site. (B) Diagrammatic representation of the YGR031W locus, indicating YGR031C-A/NAG1 nested antisense to YGR031W on the opposite strand. The circle inset indicates the exact transposon insertion identifying NAG1. nt, nucleotides; AA, amino acids; Chr., chromosome. (C) β-Galactosidase assay of the Nag1p-β-galactosidase (Nag1p-β-gal) protein product; the Nag1p chimera consists of the N-terminal 52 amino acids (AA) of Nag1p, the Tn3L and loxR sequences, and β-galactosidase from the second amino acid onwards. For comparison, the β-galactosidase level from a transposon insertion in noncoding DNA (chromosome XV, coordinate 216878) is indicated. OD, optical density. (D) Western blot identifying Nag1p tagged at its carboxy terminus with three copies of the HA epitope (3xHA). Protein extract from an untagged strain is also included as a control; both extracts were prepared from cells undergoing vegetative growth. The levels of Pgk1p, 3-phosphoglycerate kinase, were analyzed to ensure comparability between protein samples. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blot.

Nag1p localizes to the S. cerevisiae cell periphery, distinct from the YGR031WP protein product, and exhibits properties consistent with those of a plasma membrane protein. (A) Fluorescence microscopy of a Nag1p-vYFP chimera expressed from its native promoter under conditions of vegetative growth. The Spo7p-RFP chimera serves as a marker for the nuclear envelope and endoplasmic reticulum (middle image), and yeast cell morphology was visualized by differential interference contrast (DIC) microscopy (right image). (B) Fluorescence microscopy of YGR031WP-vYFP indicates its localization to the mitochondria. The MitoFluor stain was used to confirm colocalization with mitochondria (middle image), and a DIC image is provided (right). (C) Diagram illustrating predicted Nag1p transmembrane domains. Each putative transmembrane (TM) segment is shaded gray in the Nag1p schematic, and the primary sequence of each segment and its amino acid coordinates are indicated. (D) Western blots indicating the presence of Nag1p-3×HA in the pellet fraction (P13) after centrifugation of total cell lysates (T) at 13,000 rpm (16,000 × g). The known plasma membrane protein Pma1p was also found in the P13 fraction, as indicated by Western blotting with antibody directed against native Pma1p (α-Pma1p). For a further control, we used antibody directed against 3-phosphoglycerate kinase, α-Pgk1p, to confirm the presence of this protein in the supernatant fraction (S13) following centrifugation.

Nag1p production is dependent upon the MAPK Slt2p and the transcription factor Rlm1p. (A) Simplified overview of the yeast Slt2p MAPK cell wall integrity pathway. Rlm1p acts as a key transcriptional regulator downstream of Slt2p. P, phosphate. (B) β-Galactosidase assays of the Nag1p-β-galactosidase chimera during vegetative growth and in response to calcofluor white treatment in strains deleted for SLT2 and RLM1, respectively. The change from the wild-type β-galactosidase level is presented for each mutant under the indicated growth conditions. All β-galactosidase assay results were normalized per yeast cell optical density unit.

NAG1 is conserved as a unit with the YGR031W ORF in bacteria and fungi. (A) Amino acid sequence of Nag1p as predicted by conceptual translation. (B) Putative NAG1 orthologs from prokaryotic and fungal species. A schematic diagram illustrating conserved regions within the identified set of putative NAG1 orthologs is shown at the top. Each line represents the full length of the orthologous sequence; gaps in the multiple-sequence alignment are indicated as such in the figure. The prokaryotic and fungal species include Saccharomyces cerevisiae (S.cer), Kluyveromyces lactis (K.lac), Ashbya gossypii (A.gos), Vanderwaltozyma polyspora (V.pol), Candida glabrata (C.gla), Pichia guilliermondii (P.gui), Debaryomyces hansenii (D.han), Neurospora crassa (N.cra), Erwinia carotovora (E.car), Propionibacterium acnes (P.acn), and Haemophilus ducreyi (H.duc). The inset rectangle highlights the most strongly conserved region of the alignment. Identical residues are indicated as white on black, similar residues are shown as black on gray, and gaps introduced to maximize alignment are indicated by dashes. (C) Since Nag1p has not been recognized previously as a protein, neither its sequence nor the sequence of any ortholog is present in a protein database; thus, the Nag1p amino acid sequence was searched against a six-frame translation of genomic DNA sequence. The coordinates of each putative orthologous gene are indicated here, relative to each indicated database accession ID. In each instance, putative orthologs of NAG1 are found opposite orthologs of YGR031W; thus, the nested organization of NAG1 relative to YGR031W is conserved as an evolutionary unit. nt Seq., nucleotide sequence.

NAG1 contributes to yeast cell wall biogenesis. (A) The nag1-1 point mutation introduces a nonsense mutation at codon 41, without altering the predicted amino acid sequence of the YGR031WP protein product. (B) DNA microarray analysis of the nag1-1 mutant under conditions of vegetative growth. Details of this experimental design are presented in Materials and Methods. The total number of differentially expressed genes in the nag1-1 mutant relative to the wild-type (WT) strain is indicated. Genes associated with the GO cellular process term 7047 (cell wall organization/biogenesis) were statistically enriched in the set of genes with decreased transcript levels in nag1-1; these genes are shown under this GO term in the middle box. Genes associated with the GO cell component term 5886 (plasma membrane) were statistically enriched in the total set of genes identified by microarray analysis (both with increased and decreased transcript levels); these genes are also shown below the GO term. The majority of these cell surface-related genes are indicated in the heat map to the left with corresponding t statistics and P values. The full set of differentially expressed genes in the nag1-1 mutant is presented as supplementary data (see file SF1 in the supplemental material). (C) The nag1-1 mutant is sensitive to calcofluor white (CFW). For the assay shown here, we introduced a plasmid carrying the nag1-1 allele in the YGR031W genomic DNA locus into a strain of the W303 genetic background deleted for YGR031W. The resulting nag1-1 mutant was hypersensitive to calcofluor white, with increased severity at elevated temperature (37°C). SC-URA, synthetic complete medium lacking uracil. (D) Nag1p protein levels are increased upon calcofluor white (CFW) treatment, as evidenced by increased β-galactosidase activity from a Nag1p-β-galactosidase (Nag1p-β-gal) chimera.