Characterization of the sterol-binding domain of oxysterol-binding protein (OSBP)-related protein 4 reveals a novel role in vimentin organization.
Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4; also designated OSBP2 and HLM) are implicated in sterol-transport and/or sensing via binding to protein partners. The aggregation of vimentin by an N-terminal-truncated variant of ORP4 (ORP4S), but not full-length ORP4L, suggested a functional interaction with this intermediate filament. Herein, we identify ORP4 domains that interact with vimentin, and determine how sterols and OSBP influence this activity. In CHO cells, ORP4L co-localized with filamentous vimentin but extensive remodeling of vimentin filaments required mutation of a leucine repeat motif (amino acids 361-382) adjacent to the oxysterol-binding domain. Similarly, the absence of the leucine repeat in ORP4S 418-878 resulted in co-localization with aggregated vimentin filaments, suggesting that both the sterol-binding domain and leucine repeat are involved. Transient expression of OSBP leucine repeat mutants also promoted vimentin aggregation by a mechanism involving heterodimerization with ORP4L. Glutathione S-transferase (GST)-ORP4 380-878 bound vimentin, cholesterol and 25-hydroxycholesterol in vitro. However, sterol-binding or a mutation that ablated sterol-binding did not influence the interaction of GST-ORP4 with vimentin. Thus the sterol-binding domain of ORP4 binds vimentin, cholesterol and oxysterols, and interacts with the filamentous vimentin network.
Wyles
Jessica P
JP
The Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Perry
Ryan J
RJ
Ridgway
Neale D
ND
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D006888:Hydroxycholesterols / Q000378:metabolism; D008969:Molecular Sequence Data; D011987:Receptors, Steroid; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014746:Vimentin / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D002352:Carrier Proteins; D006888:Hydroxycholesterols; D011987:Receptors, Steroid; D013261:Sterols; D014746:Vimentin; D000098825:Oxysterol Binding Proteins; D002784:Cholesterol