Evidence for a dynamic and transient pathway through the TAT protein transport machinery.
Tat systems transport completely folded proteins across ion-tight membranes. Three membrane proteins comprise the Tat machinery in most systems. In thylakoids, cpTatC and Hcf106 mediate precursor recognition, whereas Tha4 facilitates translocation. We used chimeric precursor proteins with unstructured peptides and folded domains to test predictions of competing translocation models. Two models invoke protein-conducting channels, whereas another model proposes that cpTatC pulls substrates through a patch of Tha4 on the lipid bilayer. The thylakoid system transported unstructured peptide substrates alone or when fused to folded domains. However, larger substrates stalled before completion, some with amino- and carboxyl-folded domains on opposite sides of the membrane. The length of the precursor that resulted in translocation arrest (20 to 30 nm) exceeded that expected for a single 'pull' mechanism, suggesting that a sustained driving force rather than a single pull moves the protein across the bilayer. Three different methods showed that stalled substrates were not stuck in a channel or even associated with Tat machinery. This finding favors the Tha4 patch model for translocation.
Cline
Kenneth
K
Horticultural Sciences Department and Plant Molecular and Cellular Biology, University of Florida, Gainesville FL, USA. kcline@ufl.edu
McCaffery
Michael
M
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A