Key Concepts of Section 14.1

Techniques for Studying the Secretory Pathway

• All assays for following the trafficking of proteins through the secretory pathway in live cells require a way to label a cohort of secretory proteins and a way to identify the compartments where the labeled proteins are subsequently located.

• Pulse labeling with radioactive amino acids can specifically label a cohort of newly made proteins in the ER. Alternatively, a temperature-sensitive mutant protein that is retained in the ER due to misfolding at the nonpermissive temperature will be released as a cohort for transport when cells are shifted to the permissive temperature.

• Transport of a fluorescently labeled protein along the secretory pathway can be observed by microscopy (see Figure 14-2). Transport of a radiolabeled protein is commonly tracked by following compartment-specific covalent modifications to the protein.

• Many of the components required for intracellular protein trafficking have been identified in yeast by analysis of temperature-sensitive sec mutants defective for the secretion of proteins at the nonpermissive temperature (see Figure 14-4).

• Cell-free assays for intercompartmental protein transport have allowed the biochemical dissection of individual steps of the secretory pathway. Such in vitro reactions can be used to produce pure transport vesicles and to test the biochemical function of individual transport proteins.