Allan Michaels

The chloroplast 32 kDa protein is synthesized on thylakoid-bound ribosomes in Chlamydomonas reinhardtii

A cloned cpDNA fragment containing a portion of the gene for the 32–36 kDa thylakoid protein of Chlamydomonas (polypeptide D‐1) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chlamydomonas showed that the mRNA for D‐1 is bound to thylakoid membranes. Run‐off translation of thylakoid‐bound polysomes (rough thylakoids) with [35S]‐methionine yields polypeptide D‐1 as the major product. Peptide mapping with S. aureus V‐8 protease of D‐1 synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non‐polyadenylated RNA showed that D‐1 is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids.

Synthesis of a chloroplast membrane polypeptide on thylakoid-bound ribosomes during the cell cycle of Chlamydomonas reinhardii 137+

Abstract An in vitro protein synthesizing system consisting of thylakoid-bound polysomes supplemented with an S-100 from Escherichia coli has been used to investigate chloroplast membrane protein synthesis in synchronous cultures of Chlamydomonas reinhardii . The cell-free system produces at least one thylakoid membrane protein, D-2, on the basis of electrophoretic mobility and peptide mapping. Rough thylakoid membranes isolated in the middle and late light portion of the cell cycle synthesize substantial amounts of D-2 in vitro while the synthesis of D-2 was not detected by rough thylakoids isolated early in the light period or midway through the dark portion of the cell cycle. The same result was found when the synthesis of D-2 was investigated in vivo by pulse-labeling synchronous cell cultures with [ 3 H]arginine. This provides further evidence for the faithful synthesis of polypeptide D-2 in vitro. These results show that one function of thylakoid-bound ribosomes is to synthesize thylakoid membrane proteins. The results also show the cell cycle regulation of membrane protein synthesis that exists within the chloroplast on thylakoid-bound ribosomes.

In vitro synthesis and assembly of the peripheral subunits of coupling factor CF1 (α and β) by thylakoid-bound ribosomes

Abstract Bispecific antisera were prepared to a mixture of thylakoid membrane polypeptides 4.1 and 4.2. The identity of these polypeptides as the α and β subunits of coupling factor 1 (CF 1 ) was established based on the cross-reactivity of the antisera toward CF 1 from peas and by an analysis of the thm-24 mutant of Chlamydomonas which lacks the CF 1 ATPase. Photochemical labeling of thylakoid membranes with hydrophobic and hydrophilic fluorescent probes indicated that these polypeptides did not significantly penetrate the membrane bilayer. Immunoprecipitation of the translation products of thylakoid-bound and soluble ribosomes showed the thylakoids to be the major site of synthesis of the polypeptides. Immunoprecipitation of the products of translation of total cellular RNA in a reticulocyte lysate showed no evidence for substantially higher molecular weight precursors. Further analysis of the thylakoid-bound synthesis of α and β revealed that some of the in vitro synthesized polypeptides had been incorporated into the CF 0 CF 1 complex based on their release from membranes with trypsin and copurification with the CF 0 -CF 1 ATPase.

Chlorophyll antenna proteins of photosystem I: Topology, synthesis, and regulation of the 20-kDa subunit of Chlamydomonas light-harvesting complex of photosystem I

The light-harvesting complex of photosystem I (LHCI) was isolated from wild-type cells of Chlamydomonas reinhardtii; the Chl a/b-protein complex contains four major polypeptides of approximately 27, 26, 24, and 20 kDa (polypeptides 14, 15, 17.2, and 22, respectively, in the nomenclature for Chlamydomonas thylakoid proteins). Antiserum against the 20-kDa subunit of LHCI was prepared and used to determine the membrane topology, subcellular site of synthesis, and cell-cycle regulation of this polypeptide. The results indicate that the 20-kDa subunit as well as the other major LHCI polypeptides are integral membrane proteins. Moreover, protease digestion experiments reveal that the 20-kDa polypeptide is completely protected by the membrane bilayer but the 27- and 26-kDa LHCI polypeptides are exposed at the membrane surface. In vivo synthesis of the 20-kDa polypeptide is sensitive to cycloheximide but not to chloramphenicol; the form of the polypeptide recovered from in vitro translations of polyadenylated RNA is approximately 24 kDa, 4 kDa larger than the mature polypeptide. It is concluded that this LHCI polypeptide is nuclear encoded and synthesized in the cytoplasm as a higher molecular weight precursor. Synthesis of the 20-kDa polypeptide is restricted to the light period in light-dark synchronized cells. Translatable mRNA for this polypeptide accumulates during the light but levels are dramatically reduced during the dark period. Thus, synthesis of the 20-kDa subunit of LHCI appears to be transcriptionally regulated during the cell cycle.

Isolation, fractionation and analysis of intact, translatable RNA from walled algal cells

The isolation of intact, biologically active nucleic acids from wild-type Chlamydomonas reinhardtii is difficult and the cell wall mutant CW-15 is usually used for such purposes. We are investigating the expression of photosynthetic protein genes during synchronous growth of Chlamydomonas and wish to retain the use of wild-type cells in all experiments. We were not able to synchronize CW-15 under conditions used for synchronizing wild-type cells. Therefore, we have developed an efficient procedure for isolating RNA from walled cells of Chlamydomonas. The procedure is effective on cells at all stages of the cell cycle. We have also used this procedure to isolate translatable RNA from walled cells of Chlorella. With prudent choices of tissue preparation and cell breakage it should prove to be more generally useful.

Isolation and characterization of the mitochondrial DNA from the Florida spiny lobster, Panulirus argus

1. Mitochondria and mitochondrial DNA were isolated from the digestive gland of Panulirus argus. 2. The mitochondrial DNA has an average contour length of 5.13 microns which corresponds to a molecular weight of 10.10 X 10(6) daltons. 3. The molecular weight based on agarose gel electrophoresis of restricted individual DNA samples ranges from 10.04 to 10.4 X 10(6) daltons. 4. Restriction endonuclease analysis with Bam H1 and Eco R1 demonstrate variation in nucleotide sequence between individual lobsters.

Respiratory and calcium transport properties of spiny lobster hepatopancreas mitochondria

Mitochondria were isolated from the hepatopancreas of the Florida spiny lobster Panulirus argus using a high osmolarity medium containing 600 mM mannitol, 83 mM sucrose, 5 mM 4-morpholinepropanesulfonic acid, pH 7.6, 0.5% bovine serum albumin (BSA), and 1 mM EDTA. O2 uptake and Ca2+ transport were measured by electrode methods in similar media (plus 4 mM KPi, 3.3 mM MgCl2, and 0.67 mg/ml BSA, with 80 mM KCl replacing a portion of the osmotic support). Substrate-supported respiration was observed to be coupled to phosphorylation of ADP or uptake of Ca2+ ions. State 3 rates (nanogram atoms O X minute-1 X milligram protein-1 +/- SEM (N)) were: 49.2 +/- 3.9 (19), succinate; 30.9 +/- 3.9 (6), DL-palmitoyl carnitine; 29.0 +/- 2.7(9), L-malate; 40.0 +/- 2.3(3), L-glutamate; 27.7 +/- 2.2(5), D-3-hydroxybutyrate; and 26.4 +/- 2.4 (18), L-proline +/- pyruvate. alpha-Glycerol phosphate was not oxidized. Ca2+ uptake driven by succinate oxidation proceeded with Ca:O ratios of 4.0 +/- 0.2 (SEM). Hepatopancreas mitochondria were not uncoupled by Ca2+ uptake in excess of 1100 ng atoms X mg protein-1. Ca2+ efflux could be induced by ruthenium red, indicating the presence of an active Ca2+ cycle. These mitochondria may provide a favorable model system in which to study regulation of the Ca2+ cycle.

Tryptic digestion of membrane proteins synthesized on chloroplast ribosomes in Chlamydomonas reinhardtii

Chloroplast membranes of the green alga Chlamydomonas reinhardtii have been isolated from cells incubated in the presence of cycloheximide. The proteins produced under these conditions are formed on chloroplastic ribosomes. Fluorography of the polypeptides separated on polyacrylamide gels shows ten major protein species associated with the membranes that are produced on chloroplast ribosomes. These membranes were subjected to differential proteolysis. The change in electrophoretic mobility of the membrane proteins after controlled digestion indicates that proteins of molecular weight 55 000, 43 000 and 30 000 are either buried in the membrane or protected by association with other proteins. Most, if not all, of the chloroplast-synthesized thylakoid proteins contain trypsin-sensitive sites accessible from the stromal (out)-side of the isolated membrane vesicle. Thus, most chloroplast-synthesized membrane proteins are asymmetrically distributed in the thylakoid.