Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.
The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.
Karamyshev
Andrey L
AL
Department of Medical Biochemistry and Genetics, Texas A & M University System Health Science Center, College Station, 77843-1114, USA.
Johnson
Arthur E
AE
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport; D012329:RNA, Bacterial / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins