Fig 2. Carbonic anhydrase 6 is active in flagella.

A) Schematic presentation of the CAH6 gene and the insertion in the cah6 mutant. The insertion of the APHVIII cassette caused a deletion (indicated by a red crossed rectangle) of 224 bp encompassing the start codon and first exon of CAH6. The positions of the primers (F1, R1, and R2) used to track the mutation by PCR are indicated (see S1A Fig). B) Western blot analysis of isolated flagella from wild-type (g1) and the cah6 mutant were probed with anti-CAH6. Antibodies to the IFT particle protein IFT81 were used to control for equal loading. C) ¹⁸O-exhange measurements (open circles) and models (solid lines) for control (g1) and cah6 flagella. Three ¹³CO₂ isotopologues are tracked and analyzed: ¹³C¹⁶O¹⁶O (blue circles), ¹³C¹⁸O¹⁶O (green circles) and ¹³C¹⁸O¹⁸O (red circles). After determining the background rate of exchange (before time 0), isolated flagella from the strain indicated were added at time 0 and the exchange rate was measured for 400 seconds. CA activity is indicated by the accelerated conversion of ¹³C¹⁸O¹⁸O into ¹³C¹⁸O¹⁶O and ultimately ¹³C¹⁶O¹⁶O. The best fit line (k_cf) of the model after addition of isolated flagella is shown. D) Flagellar and whole cell CA activity (k_cf/k_uf) of g1 and cah6; the values were normalized for the protein concentration (mg/mL) in the respective samples and are based on repeat measurements of the same samples (n = 1). E) Western blot analysis for CAH6 in wild-type (g1) flagella. Cells were treated for 24 hours prior to the flagellar isolation as follows: The cultures were either aerated with high CO₂ (0.5%) or maintained without aeration both in the presence (+) or absence (-) of cycloheximide. Anti-IC2 was used as a loading control.