Integration of early light‐inducible proteins into isolated thylakoid membranes

Abstract

An in-vitro system has been established to study the integration of early light-inducible proteins (ELIP) into isolated thylakoid membranes. The in-vitro-expressed ELIP precursor proteins exist in two forms, a high-molecular-mass aggregate which is accessible to trypsin but no longer to the stromal processing protease and a soluble form which is readily cleaved to the mature form by the stromal protease. The mature form of ELIP is integrated into thylakoid membranes; its correct integration can be deduced from the observation that the posttranslationally transported products and the invitro integrated ELIP species are cleaved by trypsin to products of the same apparent molecular mass. Trypsin-resistant fragments of high-molecular-mass and low-molecular-mass ELIP appear to have the same size. The processed ELIP species, as well as an engineered mature form of ELIP, are integrated into isolated thylakoid membranes. Integration of the mature protein occurs in the absence of stroma, into sodium-chloride-washed, and trypsin-treated thylakoid membranes. The process of integration is almost temperature independent over 0 -30 "C. Analysis of the time course ofintegration leads to the conclusion that, under in-vitro conditions, processing but not integration into membranes is the rate-limiting step. In the absence of stroma, the ELIP precursor is bound to the thylakoid membranes, however, it is no longer accessible to the stromal maturating protease when added after binding has occurred. In conclusion, integration of ELIP differs in many essential details from that of its relatives, the light-harvesting chlorophyll u/b protein family.Early light-inducible proteins (ELIP) are short lived, thylakoid-membrane-located proteins whose genes are transiently transcribed after illumination of etiolated pea [l] or barley plantlets [2, 31. Whilc, to date, only one protein has been found in pea [l, 41, two small multigene families of different size have been characterized in barley [2].The expression of ELIP genes is not restricted to etiolated plantlets as their genes are transcribed in green plants early in the morning and under the control of the circadian clock [5]. Both in vivo [6] and after in-vitro import into intact chloroplasts [I, 71, ELIP species are integrated into the thylakoid membranes. By means of detergent fractionation of cross-linked thylakoid membranes, we found that ELIP species are associated with a preparation of photosystem-I1 (PS 11) particles [7, 81 and, in particular, with the D1 protein.D1, as a constituent of PS 11, has been found prevalently in the grana thylakoid membrane fraction [9], while a considerable portion of ELIP is obtained from the stroma thylakoid membranes [6, 71 and the intermediate membrane preparations [7]. These findings indicate that the association of EIJP with PS I1 should be confined to the site of insertion of preDl into the PS I1 particles [lo].The turnover and, consequently, the integration of D1 is accelerated after damage of PS I1 reaction centers due to highlight stress, a...

{Stable Insertion of the Early Light-induced Proteins into Etioplast Membranes Requires Chlorophyll a

“…In contrast, pElips were extracted from the membranes, both with NaOH and Na 2 CO 3 , which proved that the presence of pElips in the membrane was a result of unspecific association of these proteins with the membrane surface rather than a specific binding. A similar nonspecific association of pElips with membranes was reported to occur during the insertion of Elips into isolated thylakoid membranes (6).…”

“…Trypsin treatment of plastid membranes was performed at a protein concentration of 100 mg of protein/ml and 80 g/ml trypsin for 30 min on ice as described (6). As a control, an aliquot of the membrane suspension was incubated under the same experimental conditions in the absence of trypsin.…”

“…Prior to insertion into the thylakoid membranes pElips are processed to their mature forms by a stromal processing peptidase (5), which cleaves the N-terminal leader sequence (6). A spontaneous insertion mechanism into thylakoid membranes has been reported for Elips in barley (6) and Arabidopsis thaliana (7).…”

“…Preparation of Radioactively Labeled pElip-The low molecular mass Elip clone HV60 (4, 6) was used for in vitro transcription and translation as described (6). Translation mixtures were diluted 3-5-fold with plastid suspension buffer (50 mM Hepes/KOH, pH 8.0, 330 mM sorbitol, and 8 mM methionine) and centrifuged for 30 min at 200,000 ϫ g at 4°C.…”

Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation.

“…ELIPs, which appear to be closely associated with PSII (9,31), accumulate in mature pea leaves subjected to high light; the most pronounced induction is in high-fluence blue/UV-A radiation. An ELIP from Dunaliella increases in abundance when the alga is exposed to high light (7) and may be a zeaxanthin-binding protein (32 Increased expression from hliA in high light suggests a role for HLIP in the acclimation of cyanobacteria to phototoxic conditions.…”