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When Chlamydomonas reinhardtii cells are transferred to limiting CO,, one response is the induction of a C0,-concentrating mechanism (CCM) with components that remain t o be identified.Characterization of membrane-associated proteins induced by this transfer revealed that synthesis of the 21-kD protein (LIP-21) was regulated at the level of translatable message abundance and correlated well with the induction of CCM activity. Phase partitioning of LIP-21 and the previously characterized LIP-36 showed that both appeared to be peripherally associated with membranes, which limits their potential to function as transporters of inorganic carbon. Ultrastructural changes that occur when cells are transferred t o limiting CO, were also examined t o help form a model for the CCM or other aspects of adaptation to limiting CO,. Changes were observed in vacuolization, starch distribution, and mitochondrial location. The mitochondria relocated from within the cup of the chloroplast t o between the chloroplast envelope and the plasma membrane. In addition, immunogold labeling demonstrated that LIP-21 was localized specifically t o the peripheral mitochondria. These data suggest that mitochondria, although not previously incorporated into models for the CCM, may play an important role i n the cell's adaptation t o limiting CO,.

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...

Several changes occur in wild-type Chlamydomonas reinhardtii upon exposure to limiting CO2, including induction of several polypeptides. Polypeptide induction was previously shown to correlate with appearance of the active CO2-concentrating mechanism (CCM) of this alga. In this paper induction of polypeptides by limiting CO2 was investigated in mutants with lesions in the CCM. Mutants with lesions in the ca-1 and pmp-1 loci exhibited alterations in polypeptide induction, but it was concluded that the alterations probably do not represent their primary genetic lesions. Other changes that occur in this alga in response to limiting CO2 were also investigated. Based on a lack of significant change in the transcript abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunit genes in the wild type, it was concluded that the previously reported transient decline in synthesis of both subunits is controlled at the translational level. A transient increase in the activity of the photorespiratory enzyme phosphoglycolate phosphatase was observed in the wild type but not in a mutant, cia-5, that lacks induction of the CCM. In addition, changes in expression of genes encoding periplasmic carbonic anhydrase, a 36-kDa membrane-associated protein and a chlorophyll-binding protein occurred in the wild type but not in cia-5 in response to limiting CO2. The absence of these changes in cia-5 was attributed to a lack of either the signal itself or transduction of the signal responsible for adaptation to limiting CO2, which led to speculation that a larger range of responses are regulated by the same signal than was previously recognized. Key words: photosynthesis, photorespiration, algae, inorganic carbon transport, transcription, translation.

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