Pulse-chase analysis for studies of MHC class II biosynthesis, maturation, and peptide loading

Abstract

Pulse-chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval ("pulse"), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times ("chase"), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse-chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.

Fig.1. Pulse/chase analysis of DR molecules in DM-expressing and non-expressing B-LCL

The DM-deficient cell line, 9.5.3, its wild-type progenitor, 8.1.6, and 9.5.3 cells transfected with DM (9.5.3-DM) were pulse-labeled for 1 hr with [³⁵S]-Cys/Met and chased for 0, 2, or 8 hrs. NP-40 cell extracts (3×10⁶ cell equivalents/lane) were immunoprecipitated with the anti-DR mAb L243 and analyzed by 12% SDS-PAGE. The positions of DR α and β chains and of a 10 kDa SLIP fragment of Ii are indicated. Levels of DR-associated CLIP are substantially diminished in DM-expressing 8.1.6 cells at 2 hr and 8 hr of chase, and even further reduced when DM levels are increased by DM transfection of 9.5.3 cells. Also note inefficient IP of immature DR (mostly Ii-associated) in pulse-labeled cells with L243, which accounts for the weak bands at 0 hr chase [53].

Fig.2. Pulse/chase analysis of DQ1 molecules in DM-expressing and non-expressing B-LCL

2.2.93 and 2.2.93-DM cells were pulsed for 1 hr with [³⁵S]-Cys/Met and chased for the indicated time periods. Aliquots of cell lysates, normalized for counts, were immunoprecipitated with SPVL-3 for DQ1 and then analysed by SDS-PAGE [44]. This figure illustrates the preferential recognition of a mature confirmation of DQ1 by SPVL3, which is likely peptide-dependent and is significantly increased in the presence of DM.