Assembly-dependent trafficking assays in the detection of receptor-receptor interactions.
Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.
Margeta-Mitrovic
Marta
M
Department of Pathology, Box 0102, University of California San Francisco, 94143-0102, USA. margeta@itsa.ucsf.edu
D000818:Animals; D019556:COS Cells; D003584:Cytological Techniques / Q000379:methods; D004721:Endoplasmic Reticulum / Q000502:physiology*; D000939:Epitopes / Q000502:physiology; D009865:Oocytes; D021382:Protein Sorting Signals / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D014982:Xenopus laevis
D000939:Epitopes; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface