Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts

Abstract

We have detected a proteolytic mechanism in chloroplasts that selectively and rapidly degrades the imported small subunit of ribulose 1,5-bisphosphate carboxylase when pools of the chloroplast-synthesized large subunit are depleted. This degradation system is constitutively present and appears to be responsible for precise stoichiometric accumulation of the two subunits of the enzyme. We believe similar proteolytic mechanisms participate in regulating the accumulation of other photosynthetic proteins during chloroplast biogenesis.Ribulose 1,5-bisphosphate carboxylase/oxygenase [RbuP2Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the major protein in C3 plants, is localized in the soluble fraction of chloroplasts, where it catalyzes the initial steps in CO2 reduction (1, 2). The RbuP2Case holoenzyme consists of eight copies of each of the 52,000-to 56,000-dalton large subunit and the 12,000-to 15,000-dalton small subunit. Much is known concerning the mode of synthesis of the large subunit within chloroplasts and of the nuclear-encoded small subunit in the cytoplasm. However, many aspects of the regulation of synthesis and the pathway for subunit assembly remain to be elucidated. In particular, the mechanism(s) by which stoichiometric amounts of large and small subunits are produced during chloroplast biogenesis have not been established. In this paper, we provide evidence that, when the small subunit is produced in excess of the large, it is selectively and rapidly degraded by a proteolytic activity that is constitutively present in chloroplasts. MATERIALS 43 Ci/mg at 100% isotopic enrichment; 1 Ci = 37 GBq; ICN Chemical and Radioisotope Division) was added to a final concentration of 1 mCi/ml. The chase periods were begun on addition of a 1/10th vol of 100 mM Na2SO4. At intervals, 100-,ul aliquots were transferred to 900 ul of 100% acetone at 0°C to terminate protein synthesis and degradation. Electrophoresis. Acetone precipitates were dissolved in 2% NaDodSO4/60 mM Tris HCI, pH 8.6/60 mM dithiothreitol/5 mM E-aminocaproic acid/i mM benzamidine/15% (wt/vol) sucrose, and 5-1Al aliquots were removed for radioactivity determination (4). Equal amounts of labeled protein were subjected to electrophoresis in 10-20% (wt/vol) gradient polyacrylamide gels prepared in the buffer system of Laemmli (5). For two-dimensional electrophoresis, the precipitates were dissolved in 2% Ampholines (pH 3.5-10; LKB)/2% Nonidet P-40 (Particle Data Laboratories)/10 mM dithiothreitol/9.5 M urea/10 mM methylamine and subjected to nonequilibrium pH gradient electrophoresis for 1,600 V-hr as described (6), except that the formulation of ampholytes for a pH range of 3.5 to 9.5 (7) was used. Electrophoresis in the second dimension was carried out in the NaDodSO4/polyacrylamide gradient gels described above. Gels were treated for fluorography (8) before exposure to Kodak X-Omat AR film at -70°C. RESULTSTo study the in vivo events involved in the synthesis of RbuP2Case, we have used Chlamydomonas in pulse-chase ...

Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants.

“…In vivo labeling of membrane proteins in the presence of cycloheximide, SDS-PAGE, and fluorography were performed as described previously (Schmidt and Mishkind, 1983) except 32SOi-was used at 0.5 mCi/mL. Labeling was terminated and cells were fractionated into membrane and soluble components as described by Jensen et al (1986).…”

Synthesis and Turnover of the Chloroplast Coupling Factor 1 in Chlamydomonas reinhardi

“…Schmidt and Mishkind (27) Figure 2. The extraction of CF, from broken cells was quantitative and independent of Chl concentration during the extraction (0.5-2.0 mg Chl/ml) and ofthe volume of material extracted (0.5-3.0 ml).…”

Nuclear Gene-Regulated Expression of Chloroplast Genes for Coupling Factor One in Maize

“…However, it could as well be a consequence of overall reduced metabolic activity in the chloroplasts, some sort of feedback on transcription or some change in rates of degradation of these mRNAs. The best documented case of instability of chloroplast gene products in the absence of nuclear gene products is that of RuBP carboxylase as studied by Schmidt and Mishkind (27).…”

“…A, Chloroplast DNA (1I ig) prepared from wild-type maize (7ea mays FR 9Cms x FR 37) was digested with BamHI (B) or EcoRl (E), subjected to agarose gel (0.9%) electrophoresis with Tris-acetate buffer (19), and transferred to nitrocellulose filters (BA85, Schleicher & Schuell) after treating the gel with 0.5 N NaOH and 1.5 M NaCI and subsequently with 0.5 M Tris-HCI (pH 7.0) and 1.5 M NaCl (27). The nitrocellulose filter was prehybridized with 20 ,Ag (lanes 5 and 6) or 80 Ag (lanes 7 and 8) of hcf*-38 RNA for each lane as indicated in the figure.…”