Wiest, David L.

Most mechanisms of cell development, physiology, and signal transduction are controlled by protein-protein interactions. Immunoprecipitation of multiprotein complexes detected by flow cytometry (IP-FCM) is a means to quantitatively measure these interactions. The high sensitivity of this method makes it useful even when very little biomaterial is available for analysis, as in the case of rare primary cell subsets or patient samples. Detection of the T cell antigen receptor associated with the CD3 multiprotein complex from as few as 300 primary murine T cells is presented as an example. The method is compatible with quantitative flow cytometry techniques, making it possible to estimate the number of coimmunoprecipitated molecules. Both constitutive and inducible protein-protein interactions can be analyzed, as illustrated in related methodology using glutathione S -transferase–fusion protein pull-down experiments. IP-FCM represents a robust, quantitative, biochemical technique to assess native protein-protein interactions, without requiring genetic engineering or large sample sizes.

The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development.