b-Carbonic Anhydrases in Fungi Figure 1. Multiple sequence alignment of the zinc coordinating region from fungal b-class CAs. (A) ClustalX alignment was created using the following sequences: Sma1 [S. macrospora, Accession No. FM878639], Sma2 [FM878640], Sma3 [FM878641], Ncr1 [Neurospora crassa, Q7S631], Ncr2 [Q7S4J8], Ncr3 [Q8X0H0], Afu1 [Aspergillus fumigatus, Q4WQ18], Afu2 [A4DA32], Afu3 [Q4WPJ0], Cne1 [Cryptococcus neoformans, Q3I4V7], Cne2 [Q30E79], Sce [Saccharomyces cerevisiae, P53615], Spo [Schizosaccharomyces pombe, O94255] and Ror [Rhizopus oryzae, RO3G_10751.1]. Conserved amino acids important for Zn2+-coordination are marked by an asterisk. Identical amino acids, which are conserved in all proteins, are shaded in black; residues conserved in at least 13 of 15 sequences are shaded in dark grey and residues conserved in at least ten sequences are shaded in light grey. Arrows indicate S. macrospora cas genes with introns are given as grey boxes. The dashed box marks the region encoding the part of the protein which was used for the alignment at the top. The coding region for the mitochondrial target sequence of CAS2 is indicated as black box. (B) Amino acid identity in % is given for all sequences in pair-wise comparisons. Percentages given are based on amino acid comparison of the conserved region shown in (A). doi:10.1371/journal.pone.0005177.g001 hyphae and a high density of mitochondria can be often found at hyphal tips [26]. To confirm the mitochondrial localization of CAS2-EGFP, we stained the hyphae with Mito-Tracker or, as a negative control; we performed a co-transformation of pCAS2-GFP and pDsRED- SKL. Plasmid pDsRED-SKL encodes a DsRED with a classical C-terminal PTS1 signal for peroxisomal matrix import [27,28]. The CAS2-EGFP fusion protein clearly co-localized with the PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5177