Assays for interaction between Rab7 and oxysterol binding protein related protein 1L (ORP1L).
ORP1L belongs to the recently described family of human oxysterol binding protein homologues. We have previously shown that ORP1L localizes to late endosomes. In this chapter we describe methods that have been used to investigate the functional link of ORP1L with the protein machinery regulating late endosomal membrane trafficking. Co-immunoprecipitation, COS cell two-hybrid, and pull-down assays were applied to demonstrate a physical interaction between ORP1L and the late endosomal small GTPase Rab7. With these methods we were able to map the Rab7-binding determinant of ORP1L to the amino-terminal ankyrin repeat region (aa 1-237) and show that the interaction is preferentially with the GTP-bound form of Rab7. Furthermore, we describe approaches based on transient transfection and confocal immunofluorescence microscopy, which were employed to study the effect of this amino-terminal ORP1L fragment on late endosome morphology. The ankyrin repeat fragment induces juxtanuclear clustering of late endosomes, dependent on an intact microtubule network. When it is coexpressed with the dominant inhibitory Rab7 mutant T22N, the clustering is inhibited, suggesting that the effect involves interaction of the fragment with active Rab7.
Marie
M
Johansson
Vesa M
VM
Olkkonen
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D013261:Sterols / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D013261:Sterols; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020691:rab GTP-Binding Proteins