The Thylakoid Lumen of Chloroplasts

Abstract

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60 -65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.The chloroplast is the photosynthetic organelle of green algae and higher plants. The chloroplast architecture comprises an envelope membrane, which encloses the soluble stroma as well as the highly specialized thylakoid membrane. The stromal compartment contains mainly the components of the Calvin cycle, which are required for the fixation of carbon dioxide. The thylakoids, on the other hand, carry out the light reactions of photosynthesis leading to the production of NADPH and ATP. The thylakoid membrane has a characteristic flat shape and is differentiated into appressed grana stacks and single non-appressed stroma-exposed lamellae. The inner surface of the thylakoid membrane encloses a narrow, continuous compartment, the lumen (1, 2). Electron microscopy studies of spinach thylakoids have suggested that the lumen is a densely packed space (3). No isolation method has so far been available for obtaining a high yield of pure thylakoid lumen. Thus, the present knowledge of the lumen from a compositional and functional point of view is fragmentary and is gathered from several independent approaches, addressing only single aspects of this compartment.By developing a technique for obtaining inside-out thylakoids, the investigation of the membrane surface of the lumenal side became possible (4). This work contributed to the discovery of the extrinsic proteins PsbO, PsbP, and PsbQ (5, 6) that bind to the lumenal side of photosystem II and are thought to stabilize the water oxidizing complex (7,8). More recent studies have shown that these subunits of photosystem II occur also as soluble lumenal proteins (9). This pool of unassembled PsbO, PsbQ, and PsbP was resistant to proteolytic degradation and was capa...

The major peptidyl‐prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20

“…A BLAST search [23] revealed that the ¢rst peptide (Fig. 3A) corresponded to the 20 amino acid peptide (SwissProt accession number P82536) obtained earlier by N-terminal sequencing of the 'thylakoid lumenal 18 kDa protein' from spinach [3]. This can be considered independent con¢rmation of the localization of TLP20 in the thylakoid lumen.…”

“…The lumenal proteins were obtained from spinach thylakoids by the method of Kieselbach et al [3], which involves extensive washing of the thylakoids before disruption of the membranes by Yeda press. No PPIase activity was detected in the washed thylakoids before membrane disruption and the activity appeared only when soluble proteins were released from the lumen.…”

“…Soluble lumenal proteins were puri¢ed according to Kieselbach et al [3]. The bu¡er used during the Yeda press treatment was changed to 20 mM Tricine pH 7.8, 5 mM MgCl 2 , 100 mM sucrose for fractionation of PPIases and to 50 mM 2-(N-morpholino)ethanesul-fonic acid pH 6.0, 50 mM NaCl, 100 mM sucrose for TLP20 puri¢-cation.…”

“…The thylakoids form a continuous membrane network enclosing an inner space designated the thylakoid lumen [3]. This cellular compartment has become increasingly interesting with the advances in understanding regulation and stress protection of the photosynthetic process.…”

Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen

“…One of these questions concerns the presence of nucleotides in such a potentially multifunctional cellular compartment as particularly suggested by indications for luminal chaperones (3,9) and ATP binding to luminal proteins (11). On the other hand, luminal preparations have been tested for presence of ATP and ATPase activity without any conclusive results (12), and none of the proteins identified through proteomics have shown to have any conventional nucleotide-binding motifs (10). Furthermore, transport of nucleotides across the thylakoid membrane has not been considered, in contrast to mitochondrial membranes, where an ATP͞ADP carrier (AAC) has been extensively studied (13,14).…”