Secretion in yeast: preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro.
We have used a cytosol-free assay in which efficient translocation and signal peptide cleavage is achieved when the affinity-purified precursor of OmpA (proOmpA) is diluted out of 8 M urea into a suspension of yeast rough microsomes. This aspect of protein targeting and transport occurs in two discernible steps: (a) in the absence of ATP and cytosolic factors, the precursor binds to the membranes but is not translocated; (b) addition of ATP results in the translocation of the bound precursor and its processing to the mature form. The binding to microsomes of radiolabeled proOmpA is saturable and inhibited by the addition of unlabeled proOmpA but not by mature OmpA or other proteins. The binding of radiolabeled prepro-alpha-factor is also effectively competed by other preproteins, but not by mature ones. Scatchard analysis showed the Kd of proOmpA to be 7.5 X 10(-9) M. Binding is most likely protein mediated as treatment of the microsomes with the protease papain was found to be inhibitory. These results represent the first functional characterization of secretory protein precursor binding to membranes. Alkylation of the microsomes with NEM, washing the membranes with urea or using membranes from the (translocation) mutant ptll at the nonpermissive temperature, did not affect binding, but did eliminate the subsequent ATP-dependent translocation. The ability to subdivide translocation into individual reactions provides a more precise means of determining the membrane components involved in this process.
Sanz
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Department of Biological Chemistry, University of California, Los Angeles School of Medicine 90024.
Meyer
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D000072235:Mating Factor; D008861:Microsomes / Q000378:metabolism; D010206:Papain / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000502:physiology; D012441:Saccharomyces cerevisiae
D001425:Bacterial Outer Membrane Proteins; D005656:Fungal Proteins; D010455:Peptides; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D000072235:Mating Factor; D000255:Adenosine Triphosphate; D010206:Papain; D005033:Ethylmaleimide