Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum.
In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, addition of chemicals, or inactivation of Sec protein function using temperature-sensitive mutants or specific antibodies. In all cases, the transport block imposed by the inhibitor is reversible, permitting assessment of the requirements for transport from each intermediate stage. Based on the differential requirements for Sec protein function, as well as the distinct kinetics and efficiency of transport from each intermediate, we demonstrate that each transport intermediate is functionally distinct.
Rexach
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Division of Biochemistry and Molecular Biology, Howard Hughes Medical Research Institute, University of California, Berkeley 94720.
Schekman
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RW
D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability; D003080:Cold Temperature; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D007700:Kinetics; D000072235:Mating Factor; D008561:Membrane Fusion; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013104:Spheroplasts; D013347:Subcellular Fractions
D010455:Peptides; D011498:Protein Precursors; D000072235:Mating Factor; D002118:Calcium