5] Pulse-labeling studies of membrane assembly and protein secretion in intact cells: M13 coat protein

Abstract

This chapter reviews the pulse-labeling studies of membrane assembly and protein secretion in intact cells. The basic procedure in such experiments is to (1) grow cells in a medium with a low concentration of at least one amino acid; (2) briefly pulse-label with that amino acid; (3) “chase” with a large chemical excess of the amino acid; (4) remove portions of the culture at various intervals to an environment that will “quench” the processes of interest ; (5) analyze topography where necessary by subcellular fractionation and proteolysis of closed, vesicular organelles; and (6) assay the pulse-labeled protein by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography or autoradiography. The cells grow in a medium with only small levels of the pulse-labeling amino acid. Uptake of labeled amino acid is rapid and its incorporation proceeds without dilution through large internal pools. The addition of a large excess of nonradioactive amino acid leads to efficient and rapid competitive dilution of the isotope incorporation. Escherichia coli have each of these characteristics of favorable pulse-labeling and the chase.