Disassembly and degradation of photosystem I in an in vitro system are multievent, metal-dependent processes.
An in vitro system was created to study the process of membrane protein degradation by using photosystem I (PS1) as a model membrane protein. Purified chloroplast membranes were incubated at 30 degrees C in a defined buffer along with various extracts or reagents to reconstitute the disassembly and degradation of PS1, which was monitored by a variety of techniques that probe the integrity of the PS1 complex: photo-biochemical assays, semi-native gel electrophoresis, low temperature fluorescence spectroscopy, and immunoblots using antibodies against different PS1 subunits. During a typical time course, degradation of PS1 appeared to be a multievent process, with disassembly of the complex preceding proteolysis of the subunits. The first change seen was a rapid (<5 min) decrease in PS1 photochemical activity. This was followed by a diminution of far-red fluorescence emission from the core antenna of PS1 and a slower disassembly of the PS1 chlorophyll-protein core complex, as visualized by semi-native gel electrophoresis. Surprisingly, the latter was not accompanied by a similar rate of proteolysis of the PsaA core subunit. In contrast, addition of soluble proteases caused rapid loss of immuno-detectable PS1 polypeptides and cleavage of the major PS1 polypeptides in interhelical loops. The in vitro degradation process was time- and temperature-dependent but did not require ATP, GTP, or soluble chloroplast proteins. Chelation of divalent cations by EDTA inhibited the later steps of disassembly and proteolysis, and this effect could be reversed by addition of micromolar Zn2+, with Co2+ and Ca2+ providing somewhat lower activity.
Henderson
J Nathan
JN
Department of Chemistry, University of Alabama, Tuscaloosa, Alabama 35487-0336, USA.
Zhang
Jianying
J
Evans
B Walter
BW
Redding
Kevin
K
D000818:Animals; D002696:Chlamydomonas / Q000378:metabolism; D002736:Chloroplasts / Q000737:chemistry; D010450:Endopeptidases / Q000494:pharmacology; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D010777:Photochemistry; D045331:Photosystem I Protein Complex / Q000737:chemistry* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature
D046911:Macromolecular Substances; D008670:Metals; D045331:Photosystem I Protein Complex; D011480:Protease Inhibitors; D010450:Endopeptidases