Expression, assay, and functional properties of RILP

Methods Enzymol. 2005;403:664-75. doi: 10.1016/S0076-6879(05)03057-0.

Abstract

Rab proteins are master regulators of vesicular membrane traffic of endocytic and exocytic pathways. They basically serve to recruit proteins and lipids required for vesicle formation, docking, and fusion. Each Rab protein is able to recruit one or more effectors, and, through the action of effectors, it drives its specific downstream functions. The Rab interacting lysosomal protein (RILP) is a common effector of Rab7 and Rab34, two Rab proteins implicated in the biogenesis of lysosomes. RILP is recruited onto late endosomal/lysosomal membranes by Rab7-GTP where it induces the recruitment of the dynein-dynactin motor complexes. Therefore, through the timed and selective dynein motor recruitment onto late endosomes and lysosomes, Rab7 and RILP control transport to endocytic degradative compartments. A similar role for Rab7 and RILP has been demonstrated also for phagosomes. Indeed, RILP recruits dynein-dynactin motors on Rab7-GTP-positive phagosomes and the recruitment not only displaces phagosomes centripetally, but also promotes the extension of phagosomal tubules toward late endocytic compartments. RILP is therefore a key protein for the biogenesis of lysosomes and phagolysosomes. This chapter describes how to express wild-type or mutated RILP in mammalian cells, and how to test the effects caused by RILP dysfunction. In particular, we report assays to monitor the interaction between RILP and Rab7, morphology and distribution of endosomes, and to measure degradation of endocytic markers.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Base Sequence
  • Carrier Proteins / genetics
  • Carrier Proteins / physiology*
  • DNA Primers
  • Genetic Vectors
  • HeLa Cells
  • Humans
  • Lipoproteins, LDL / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Organelles / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA Primers
  • Lipoproteins, LDL
  • RILP protein, human