Phosphoproteins and protein-kinase activity in isolated envelopes of pea (Pisum sativum L.) chloroplasts

A protein kinase was found in envelope membranes of purified pea (Pisum sativum L.) chloroplasts. Separation of the two envelope membranes showed that most of the enzyme activity was localized in the outer envelope. The kinase was activated by Mg2+ and inhibited by ADP and pyrophosphate. It showed no response to changes in pH in the physiological range (pH 7-8) or conventional protein substrates. Up to ten phosphorylated proteins could be detected in the envelope-membrane fraction. The molecular weights of these proteins, as determined by polyacrylamide-gel electrophoresis were: two proteins higher than 145 kDa, 97, 86, 62, 55, 46, 34 and 14 kDa. The 86-kDa band being the most pronounced. Experiments with separated inner and outer envelopes showed that most labeled proteins are also localized in the outer-envelope fraction. The results indicate a major function of the outer envelope in the communication between the chloroplast and the parent cell

The protein import apparatus of chloroplasts

Routing of cytosolically synthesized precursor proteins into chloroplasts is a specific process which involves a multitude of soluble and membrane components. In this review we wil1 focus on early events of the translocation pathway of nuclear coded plastidic precursor proteins and compare import routes for polypeptide of the outer chloroplast envelope to that of internal chloroplast compartments. A number of proteins housed in the chloroplast envelopes have been implied to be involved in the translocation process, but so far a certain function has not been assigned to any of these proteins. The only exception could be an envelope localized hsc 70 homologue which could retain the import competence of a precursor protein in transit into the organelle

A guanosine 5′-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-32P]GTP to ADP to yield (-32P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP

The major chloroplast envelope polypeptide is the phosphate translocator and not the protein import receptor

DURING photosynthetic CO2 fixation, fixed carbon is exported from the chloroplasts in the form of triose phosphate by the chloroplast phosphate translocator, which is the principal polypeptide (E29) from spinach chloroplast envelopes1. We have sequenced this nuclear-coded envelope membrane protein from both spinach and pea chloroplasts2,3. An envelope membrane protein, E30, has been identified as a possible receptor for protein import into pea chloroplasts using an anti-idiotypic antibody approach4–6; antibodies raised against purified E30 inhibited binding and import of proteins into chloroplasts7. The amino-acid sequence of E30 deduced from its complementary DNA7 turned out to be highly homologous to that of E29, assigned by us as the spinach phosphate translocator2, and was identical to the corresponding polypeptide from pea chloroplasts3. Differences in the binding properties to hydroxylapatite of £30 and the phosphate translocator suggested that E30 was not responsible for the chloroplast phosphate-transport activity but was the chloroplast import receptor7. Here we present evidence that argues against this and which identifies E30 as the chloroplast phosphate translocator

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins