Identification and characterization of two closely related β‐carbonic anhydrases from Chlamydomonas reinhardtii

Abstract

Aquatic photosynthetic organisms such as the green alga Chlamydomonas reinhardtii respond to low-CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Important components of the CCM are the carbonic anhydrases (CAs), zinc metalloenzymes that catalyze the interconversion of CO(2) and HCO(-)(3). Six CAs have previously been identified in C. reinhardtii. Here, we identify and characterize two additional beta-type CAs. These two CAs are closely related beta-type CAs and have been designated as CAH7 and CAH8. Conceptual translation shows that CAH7 and CAH8 encode proteins of 399 and 333 amino acids, respectively, and they contain targeting sequences. An unusual characteristic of these two CAs is that they have carboxy-terminal extensions containing a hydrophobic sequence. Both these CAs are constitutively expressed at the transcript and protein level. The CAH7 and CAH8 open reading frames were cloned in the overexpression vector pMal-c2x and expressed as recombinant proteins. Activity assays showed that CAH7 and CAH8 are both active CAs. Antibodies were raised against both CAH7 and CAH8, and immunolocalization studies showed that CAH8 was localized in the periplasmic space. A possible role for CAH8 in the inorganic carbon acquisition by C. reinhardtii is discussed.

Activation of the Carbon Concentrating Mechanism by CO2 Deprivation Coincides with Massive Transcriptional Restructuring in Chlamydomonas reinhardtii

“…These results are confirmed by the companion article (Fang et al, 2012), including the activation of CAH1 and CAH4/ CAH5. As a notable difference to the observations of Ynalvez et al (2008) and Fang et al (2012), we found CAH3 slightly upregulated. In the cia5 mutant, Fang et al found that CAH5, the most highly upregulated CA gene in wild-type cells, is actually repressed.…”

“…Four of the nine a-and b-CA genes (CAH1, CAH4/CAH5, and CAH7) were activated (twofold or more) (Figure 2; see Supplemental Data Sets 2 and 7 online) and none were repressed significantly (FDR # 0.01). Transcripts encoding CAH1, an a-type, periplasmic CA and CAH4/CAH5, recently duplicated, mitochondrial, b-type CAs were induced by over 2 9 (512-fold), similar to RT-PCR observations by Ynalvez et al (2008). These results are confirmed by the companion article (Fang et al, 2012), including the activation of CAH1 and CAH4/ CAH5.…”

Evolution of carbonic anhydrases in fungi

“…The multiplicity of fungal CA-isoforms leads to the assumption that the proteins are involved in various processes and are targeted to diVerent cellular compartments, as it has been described in plants, algae and animals (Hewett-Emmett and Tashian 1996; Moroney et al 2001;Supuran 2008;Ynalvez et al 2008). Because of the high degree of sequence identity, these functional speciWcations and the subcellular localization events are thought to be quite recent (Hewett-Emmett and Tashian 1996).…”

SLC4 family transporters in a marine diatom directly pump bicarbonate from seawater

“…In the freshwater chlorophyte, Chlamydomonas reinhardtii, chloroplastic CAs, a pyrenoidal component including low-CO 2 -inducible protein (LCI) B/C, and the membrane transporter, LCI1, are known to facilitate the CCM (14)(15)(16)(17). Additionally, the role of extracellular CAs (CA ext ) in CCM has been controversial in the study of green algae (18).…”