Disassembly and Degradation of Photosystem I in an in Vitro System Are Multievent, Metal-dependent Processes

Abstract

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°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 temperaturedependent 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 Zn 2؉ , with Co 2؉ and Ca 2؉ providing somewhat lower activity.

Characterization of the structural changes and photochemical activity of photosystem I under Al3+ effect

“…These changes are shown by the conversion of a-helix to b-sheet, b-antiparallel and turn structures of the polypeptides. This effect can be carried out especially at the transmembrane intrinsic polypeptides rich in a-helices such as PsaA and PsaB subunits, LHCI and other associated intrinsic polypeptides [4,6,9,65]. These conformational changes demonstrate that Al 3+ alter the protein secondary structures revealing that a reorganization of PSI domain has occurred including proteins and pigments within PSI RC and LHCI.…”

The plastid-encoded PsaI subunit stabilizes photosystem I during leaf senescence in tobacco

“…Unfortunately, not much is known about proteases involved in the degradation of PSI in higher plants. The degradation of PSI from Chlamydomonas reinhardtii was only studied in vitro after the addition of soluble proteases (Henderson et al , 2003), but it is unclear to what extent these results can be extrapolated to the in situ degradation of PSI in intact leaves of higher plants. A role for the FTSH protease in PSI biogenesis and accumulation was recently described; however, this effect was unrelated to PSI turnover and degradation (Järvi et al , 2016).…”

Self-Assembling Peptide Detergents Stabilize Isolated Photosystem Ion a Dry Surface for an Extended Time

“…Monitoring of the F685/F735 chlorophyll fluorescence ratio has been used to study many in vivo and in vitro biological processes, including (1) the effect of leaf temperature [34], (2) seasonally induced chlorophyll breakdown in trees [35], (3) herbicide phytotoxicity in algae [36], (4) high-light-induced photoinhibition [37], (5) iron deficiency in algae [38], and (6) PS-I stability and disassembly [39]. …”