Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus, as related to possible functions of carboxysomes

Abstract

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.