The properties of the positively charged loop region in PSI-G are essential for its "spontaneous" insertion into thylakoids and rapid assembly into the photosystem I complex.
The PSI-G subunit of photosystem I (PSI) is an 11-kDa membrane protein that plays an important role in electron transport between plastocyanin and PSI and is involved in the stability of the PSI complex. Within the complex, the PSI-G subunit is bound to PSI-B and is in contact with Lhca1. PSI-G has two transmembrane spans connected by a positively charged stromal loop. The loop is inaccessible to proteases, indicating a tightly bound location within the PSI complex. Here, we have studied the insertion mechanism and assembly of PSI-G. We show that the protein inserts into thylakoids by a direct or "spontaneous" pathway that does not involve the activities of any known chloroplast protein-targeting machinery. Surprisingly, the positively charged stromal loop region plays a major role in this process. Mutagenesis or deletions within this region almost invariably lead to a marked lowering of insertion efficiency, strongly indicating a critical role for the loop in the organization of the transmembrane regions prior to or during membrane insertion. Finally, we have examined the assembly of newly inserted PSI-G into the PSI complex, since very little is known of the assembly pathway for this large multimeric complex. Interestingly, we find that inserted PSI-G can be found within the full PSI complex within the import assay time frame after insertion into thylakoids, strongly suggesting that PSI-G normally associates at the end of the assembly process. This is consistent with its location on the periphery of the complex.
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 1870 Frederiksberg, Denmark.
Agnieszka
A
Zygadlo
Colin
C
Robinson
Henrik Vibe
HV
Scheller
Alexandra
A
Mant
Poul Erik
PE
Jensen
D000255:Adenosine Triphosphate / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004579:Electron Transport; D015180:Electrophoresis, Gel, Two-Dimensional; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D016296:Mutagenesis; D009711:Nucleotides / Q000737:chemistry; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D020524:Thylakoids / Q000201:enzymology / Q000378:metabolism*
D009711:Nucleotides; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011522:Protons; C074492:photosystem I, PSI-G subunit; D000255:Adenosine Triphosphate; D004247:DNA