Protein Arginine Methylation in Candida albicans: Role in Nuclear Transport

C. albicans Hmt1 is not essential and is the major PRMT. (A) hmt1Δ/hmt1Δ (AMC11; HMT1−) and hmt1Δ/hmt1Δ+HMT1 (AMC14; HMT1+) C. albicans strains were created using a PCR-based strategy. Total protein (10 μg) from these strains was analyzed by immunoblotting with anti-ScHmt1 antiserum. (B) C. albicans strains were labeled in vivo with [³H]AdoMet as described in Materials and Methods. Proteins in cell extracts (50 μg) were resolved by SDS gel electrophoresis and analyzed by Coomassie blue staining and fluorography. The dried gel was exposed to Kodak X-Omat AR film at −80°C for 10 days to detect methylated proteins. Arrows indicate major ³H-methylated polypeptides found in the hmt1Δ/hmt1Δ+HMT1 strain (AMC14; HMT1+) expressing the methyltransferase and absent or greatly reduced in the mutant hmt1Δ/hmt1Δ strain (AMC11; HMT1−). (C) Amino acid analysis of C. albicans proteins from lysed cells in vivo labeled with [³H]AdoMet. Acid-hydrolyzed proteins (150 μg) were fractionated on a cation-exchange column as described in Materials and Methods. Radioactivity (³H) (solid line) was determined by scintillation counting. Nonradiolabeled amino acid standards of ADMA, ω-MMA, and SDMA were added to the hydrolysate and detected using a ninhydrin assay (dashed line). The upper panel shows the labeling in cells expressing Hmt1 (AMC14; HMT1+), and the lower panel shows the labeling in cells lacking Hmt1 (AMC11; HMT1−). The radioactivity in the off-scale fractions of the upper panel is 2,625 for fraction 72, 5,707 for fraction 73, and 1,172 for fraction 87. In these experiments, the ³H-labeled methylated derivatives eluted slightly earlier than the nonisotopically labeled standards due to an isotope effect (18).

C. albicans Rmt2 catalyzes the formation of δ-MMA as a minor PRMT. (A) C. albicans strains RMT2/rmt2Δ (AMC28; RMT2+) and rmt2Δ/rmt2Δ (AMC30; RMT2−) were labeled in vivo with [³H]AdoMet as described in Materials and Methods. Proteins in cell extracts (50 μg) were analyzed by SDS gel electrophoresis as described in the legend to Fig. 2. The dried gel was exposed to Kodak BioMax MS film at −80°C for 10 days. (B) Amino acid analysis of C. albicans proteins from lysed RMT2/rmt2Δ and rmt2Δ/rmt2Δ cells labeled in vivo with [³H]AdoMet as described in the legend Fig. 2. The upper panel shows the labeling in cells expressing Rmt2 (AMC28; RMT2+), and the lower panel shows the labeling in rmt2Δ/rmt2Δ cells (AMC30; RMT2−). The arrow indicates the peak that decreases in the absence of Rmt2. The radioactivity in the off-scale fractions for the upper panel is 2,062 for fraction 73, 2,759 for fraction 74, and 1,079 for fraction 75; for the lower panel it is 1,973 for fraction 73 and 1,411 for fraction 74. The radioactive derivatives are expected to elute slightly earlier than the standards, as described in the Fig. 2 legend.

The RGG domain of C. albicans Npl3, but not full-length protein, can function in S. cerevisiae. (A) Clustal W alignment (9) of S. cerevisiae RNA-binding protein Npl3 (S. cer.) and the C. albicans Npl3 ortholog (C. alb.). RNA recognition motifs (RRMs) and arginine-glycine-rich (RGG) domains are shown. Sky1-mediated phosphorylation of serine 411 (bold) in the conserved C-terminal heptapeptide facilitates Npl3 nuclear import in S. cerevisiae (20, 50). SR dipeptides found in S. cerevisiae, but not C. albicans, Npl3 are underlined. Asterisks denote identical residues in all sequences, colons denote conserved substitutions, and periods denote semiconserved substitutions. (B) An S. cerevisiae strain lacking NPL3 (PSY814) was transformed with plasmids that express PrA alone (vector; pNOPPATA) or PrA fused to S. cerevisiae Npl3 (ScNpl3; pPS2389), C. albicans Npl3 (CaNpl3; pAM361), or a chimeric protein composed of the first three domains of ScNpl3 with the RGG domain of CaNpl3 (ScNpl3-CaRGG; pAM362). Cells were grown overnight to mid-log phase in medium lacking leucine and washed, and 10-fold dilutions (10⁶ to 10³ cells) were plated on 5-FOA medium and grown at 30°C for 2 days. (C) hmt1Δ (YAM533) and HMT1 (YAM535) S. cerevisiae strains expressing myc-tagged ScNpl3 were transformed with the PrA-Npl3 plasmids shown in panel B. Proteins from mid-log-phase cells were precipitated with IgG-Sepharose. Proteins isolated from 1.7 mg lysate (beads) and total lysates (20 μg) were analyzed by immunoblotting with a polyclonal anti-myc antiserum, which also binds to PrA.