A 36 Kilodalton Limiting-CO₂ Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase

Abstract

Chiamydomonas reinhardtii possesses a C02-concentrating mechanism, induced by limiting C02, which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO2, but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO2. It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A @) RNA from cells only after exposure to limiting CO2. The absence of translatable mRNA for this polypeptide in C02-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO2 induced 36 kilodalton polypeptide and the similarly sized, limiting-CO2 induced periplasmic carbonic anhydrase.Chiamydomonas reinhardtii, as well as several other green microalgae and cyanobacteria, exhibit a C02-concentrating system induced by low external levels of C02. This system (reviewed in 1, 13, and 15), which gives them a high apparent affinity for CO2, results from the active transport of inorganic carbon (C1)2. air-adapted cells), their apparent affinity for C02 is much higher than that of C02-enriched cells.Components of the C02-concentrating mechanism include a mechanism for active transport of C1, an internal carbonic anhydrase, and a periplasmic carbonic anhydrase (1, 15). Induction of the C02-concentrating mechanism and periplasmic carbonic anhydrase occur in response to low external Ci. Lack of induction in the presence of CHI indicates that de novo protein synthesis is essential (5, 12, 17). Translation of one or more proteins involved in the system must be initiated or upregulated.The best-characterized protein for which specific involvement in the system and a function have been identified is the periplasmic carbonic anhydrase. Coleman et al. (6) identified a soluble 37 kD polypeptide as the periplasmic carbonic anhydrase in Chlamydomonas. The polypeptide appears following transfer of C02-enriched cells to air conditions. It is excreted into the medium by the CW15 mutant which lacks a normal cell wall.The synthesis of other polypeptides has been correlated with the induction of the C02-concentrating mechanism, but so far none have been assigned a specific function or location. It is possible that one or more of these proteins could be involved with the transport mechanism, si...

Chlamydomonas reinhardtii thermal tolerance enhancement mediated by a mutualistic interaction with vitamin B12-producing bacteria

“…reinhardtii and bacterial strains and growth conditions Wild-type C. reinhardtii strains 21gr (cc1690) and 137c (cc125) were obtained from the Chlamydomonas Resource Center (http://chlamycollection.org/) and were grown phototrophically under continuous illumination (120 mE m À 2 sec À 1 ) in minimal salts medium or on agar (1.5%) solidified plates (Geraghty et al, 1990) in a 5% CO 2 atmosphere. Bacterial species were grown on TY (Beringer 1974) or rhizobial minimal medium (Broughton et al, 1986) (Campbell et al, 2006;Taga et al, 2007) were generously provided by Michiko Taga (University of California-Berkeley).…”

Physiological and Molecular Aspects of the Inorganic Carbon-Concentrating Mechanism in Cyanobacteria

“…Control by ambient CO2 level of the synthesis of a 36-kD polypeptide appears to be exerted via regulation of mRNA abundance. This membrane-associated polypeptide (apparently chloroplast-, but not thylakoid-located) is distinct from the periplasmic CA as shown by use of antibodies (10).…”

“…No other polypeptide has yet been shown to respond to low C02 level, although the number of carboxysomes increases (27). In Chlamydomonas, on the other hand, the levels of several polypeptides have been shown to rise during adaptation to low C02 (10,17,26), including the 37-kD (the periplasmic CA, see above). Specific functions have not yet been identified for the other polypeptides, including the 44-and 46-kD polypeptides missing in the high-C02-requiring transport mutant pmp-1.…”

Two Polypeptides in the Inner Chloroplast Envelope of Dunaliella tertiolecta Induced by Low CO2

“…This DIC pump is suppressed by elevated CO2 concentrations (1-5% CO2 in air [v/v]) and is formed in these 'high-CO2 cells" within 4 to 6 h in the light when cultures receive only air levels of CO2 ("low-CO2 cells'). During the induction of the DIC pump in Chlamydomonas, a number of proteins are formed preferentially (1,4,7,13,14,21,22), one of which is a periplasmic carbonic anhydrase (3, 16). Working models for the DIC-concentrating mechanism in green algae suggest that active DIC transporters should be membrane-bound proteins, which may be located at the plasmalemma and/or at the chloroplast envelope (23).…”

“…During the induction of the DIC pump in Chlamydomonas, a number of proteins are formed preferentially (1,4,7,13,14,21,22), one of which is a periplasmic carbonic anhydrase (3,16). Working models for the DIC-concentrating mechanism in green algae suggest that active DIC transporters should be membrane-bound proteins, which may be located at the plasmalemma and/or at the chloroplast envelope (23).…”