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1.  The Oxysterol-binding Protein Homologue ORP1L Interacts with Rab7 and Alters Functional Properties of Late Endocytic Compartments 
Molecular Biology of the Cell  2005;16(12):5480-5492.
ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.
doi:10.1091/mbc.E05-03-0189
PMCID: PMC1289395  PMID: 16176980
2.  The Two Variants of Oxysterol Binding Protein-related Protein-1 Display Different Tissue Expression Patterns, Have Different Intracellular Localization, and Are Functionally Distinct 
Molecular Biology of the Cell  2003;14(3):903-915.
Oxysterol binding protein (OSBP) homologs comprise a family of 12 proteins in humans (Jaworski et al., 2001; Lehto et al., 2001). Two variants of OSBP-related protein (ORP) 1 have been identified: a short one that consists of the carboxy-terminal ligand binding domain only (ORP1S, 437 aa) and a longer N-terminally extended form (ORP1L, 950 aa) encompassing three ankyrin repeats and a pleckstrin homology domain (PHD). We now report that the two mRNAs show marked differences in tissue expression. ORP1S predominates in skeletal muscle and heart, whereas ORP1L is the most abundant form in brain and lung. On differentiation of primary human monocytes into macrophages, both ORP1S and ORP1L mRNAs were induced, the up-regulation of ORP1L being >100-fold. The intracellular localization of the two ORP1 variants was found to be different. Whereas ORP1S is largely cytosolic, the ORP1L variant localizes to late endosomes. A significant amount of ORP1S but only little ORP1L was found in the nucleus. The ORP1L ankyrin repeat region (aa 1–237) was found to localize to late endosomes such as the full-length protein. This localization was even more pronounced for a fragment that additionally includes the PHD (aa 1–408). The amino-terminal region of ORP1L consisting of the ankyrin repeat and PHDs is therefore likely to be responsible for the targeting of ORP1L to late endosomes. Interestingly, overexpression of ORP1L was found to enhance the LXRα-mediated transactivation of a reporter gene, whereas ORP1S failed to influence this process. The results suggest that the two forms of ORP1 are functionally distinct and that ORP1L is involved in control of cellular lipid metabolism.
doi:10.1091/mbc.E02-08-0459
PMCID: PMC151568  PMID: 12631712
3.  OSBP-Related Protein 8 (ORP8) Regulates Plasma and Liver Tissue Lipid Levels and Interacts with the Nucleoporin Nup62 
PLoS ONE  2011;6(6):e21078.
We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (−34%, −26%, −37%, respectively) and liver tissue (−40%, −12%, −24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [3H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.
doi:10.1371/journal.pone.0021078
PMCID: PMC3115989  PMID: 21698267

Results 1-3 (3)