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.
Zygadlo
Agnieszka
A
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 1870 Frederiksberg, Denmark.
Robinson
Colin
C
Scheller
Henrik Vibe
HV
Mant
Alexandra
A
Jensen
Poul Erik
PE
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