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Proteasomes have long been known to mediate the degradation of polyubiquitinated proteins in the cytoplasm and the nucleus. Additionally, proteasomes have been identified as participating in cellular degradative pathways involving the endomembrane system. In conjunction with the endoplasmic reticulum, proteasomes serve as a quality control mechanism for disposing of malfolded newly synthesized proteins, while on the endocytic pathway they serve to facilitate the degradation of key signaling and nutrient receptors as well as the destruction of phagocytosed pathogens. Our laboratory has identified a direct interaction between the late endocytic Rab7 GTPase and the alpha-proteasome subunit, XAPC7, thus providing the first molecular link between the endocytic trafficking and cytosolic degradative machineries. In this chapter reagents and methods for studying the regulation and interactions between XAPC7, the 20S proteasome, and Rab7 are described.

Proteasomes have long been known to mediate the degradation of polyubiquitinated proteins in the cytoplasm and the nucleus. Additionally, proteasomes have been identified as participating in cellular degradative pathways involving the endomembrane system. In conjunction with the endoplasmic reticulum, proteasomes serve as a quality control mechanism for disposing of malfolded newly synthesized proteins, while on the endocytic pathway they serve to facilitate the degradation of key signaling and nutrient receptors as well as the destruction of phagocytosed pathogens. Our laboratory has identified a direct interaction between the late endocytic Rab7 GTPase and the alpha-proteasome subunit, XAPC7, thus providing the first molecular link between the endocytic trafficking and cytosolic degradative machineries. In this chapter reagents and methods for studying the regulation and interactions between XAPC7, the 20S proteasome, and Rab7 are described.