In the present study, we show that in HeLa cells the OSBP homologue ORP1L localizes on late endocytic compartments and its distribution overlaps largely with those of Lamp-1, the small GTPase Rab7, a central regulator of late endosomal membrane trafficking events, and Rab9, a GTPase with a key role in the route from late endosomes to the trans
-Golgi (Lombardi et al., 1993
; Carroll et al., 2001
). The finding that ORP1L overexpression induced clustering of LE/lysosomes prompted us to test whether ORP1L interacts with the late endosomal small GTPases. We show that both the endogenous and the overexpressed ORP1L and Rab7 proteins can be coimmunoprecipitated. The interaction seems to be specific for Rab7, because ORP1L does not coimmunoprecipitate with Rab9 or Rab6. Further evidence for the ORP1L–Rab7 interaction is provided by a COS cell two-hybrid assay, the results of which suggest that ORP1L preferably binds the active, GTP-bound form of Rab7. A third piece of evidence for a specific interaction between ORP1L and Rab7 is provided by an assay in which GST-Rab7 loaded with nonhydrolyzable GTPγS was capable of pulling down in vitro-translated ORP1L. This assay demonstrates that, of the truncated ORP1L fragments used, only ones that contain the N-terminal ANK repeat region, or the ANK region alone, bind Rab7-GTP, strongly suggesting that this region (aa residues 1–237) is sufficient for the Rab7 interaction. However, the present data do not indicate whether it is the ankyrin repeats themselves that specify this binding interaction.
The GTP-binding and hydrolysis cycle of Rab GTPases is connected with the dynamic localization of the proteins on specific membrane compartments (Zerial and McBride, 2001
). Therefore, the GTP cycle can be indirectly monitored by observing the intracellular distribution of the protein using FRAP technology (Jordens et al., 2001
). The FRAP analysis of endo/lysosome-associated GFP-Rab7 revealed that ORP1L substantially slows down the rate at which membrane-bound bleached Rab7 is replaced by fluorescing GFP-Rab7. This suggests that ORP1L stabilizes active GTP-Rab7 on these compartments.
The PHD is immediately downstream of the ANK region of ORP1L. Using a vesicle pull-down assay, we show that this PHD binds several phosphoinositides with low affinity and specificity. The binding properties of ORP1L are thus similar to those of a majority of PHDs studied. Typically, additional interaction determinants or oligomerization of a PHD to increase avidity are necessary for efficient membrane targeting (Lemmon and Ferguson, 2000
). The ANK region alone is capable of minimal LE targeting, consistent with the ability of this fragment to interact with Rab7. In HeLa cells, there is no significant difference in the localization of the ANK region and a fragment containing the ANK region and the PHD (ANK+PHD), so the role of the PHD in the LE targeting in this cell model remains unclear. However, in Chinese hamster ovary cells the ANK+PHD fragment showed clearly stronger LE targeting that the ANK region alone (Johansson et al., 2003
), suggesting that the ANK region and the PHD may both play a role in the membrane association of ORP1L. In this respect, ORP1L could resemble another Rab binding partner, early endosomal antigen-1, which interacts with Rab5. This protein is recruited to early endosomes via interactions with both Rab5-GTP and phosphatidylinositol-3-phosphate (Simonsen et al., 1998
; Gaullier et al., 2000
The ANK and ANK+PHD fragments of ORP1L induce clustering of Lamp-1-positive LEs/lysosomes in the juxtanuclear region, but no obvious formation of enlarged Lamp-1 and Rab7-positive structures frequently observed with the full-length protein. Our recent findings show that the enlarged structures induced upon overexpression of the full-length protein in fact represent autophagic vacuoles (unpublished observations). Expression of the dominant inhibitory mutant T22N Rab7 counteracts the clustering phenotype induced by the ORP1L fragments. This mutant Rab7 was previously shown to cause dispersion of LEs (Bucci et al., 2000
). The fact that Rab7 T22N reverses the LE clustering by ORP1L ANK and ANK+PHD indicates that this process requires active Rab7, in support of a functional interplay of ORP1L and the small GTPase. The LE/lysosomal localization of overexpressed ORP1L does not require simultaneous overexpression of Rab7, consistent with the notion that stoichiometric amounts of Rab7 are not necessary for membrane recruitment of ORP1L. This indicates that, even though ORP1L colocalizes extensively with Rab7 and interacts physically with the GTPase, its LE association involves other protein or lipid determinants in addition to Rab7.
The clusters induced by ORP1L or its N-terminal fragments are dissociated into smaller structures and dispersed throughout the cytoplasm when microtubules are destroyed using nocodazole. It has previously been demonstrated that the perinuclear localization of LEs/lysosomes as well as the transport of internalized proteins from early endosomes to LEs depends on microtubules (reviewed by Apodaca, 2001
). The present finding is consistent with this and shows that, even though the clustering of LEs induced by ORP1L, ANK, and ANK+PHD is not fully reversed by microtubule dissociation, the large clusters in the juxtanuclear region are microtubule dependent. Other Rab7 binding partners identified so far include RILP, a protein that links Rab7-positive LEs to microtubule-dependent dynein/dynactin motor complexes and has strong effects on LE/lysosomal transport (Cantalupo et al., 2001
; Jordens et al., 2001
); Rabring7, which affects epidermal growth factor degradation and causes perinuclear aggregation of lysosomes (Mizuno et al., 2003
); and the phosphatidylinositol 3′-kinase VPS34 and its adaptor protein p150 (Stein et al., 2003
). The clustering of LEs/lysosomes induced by ORP1L and its ANK and ANK+PHD fragments resembles the effects of RILP or Rabring7 overexpression or that of Rab7 itself (Bucci et al., 2000
). We show that the clustering effects of ORP1 are inhibited by overexpression of p50dynamitin, which uncouples the dynein motors from cargo (Burkhardt et al., 1997
; Valetti et al., 1999
) and that p150glued
, a component of the dynein/dynactin complex, is concentrated on ORP1-positive LEs/lysosomes. Recruitment of dynein/dynactin motor complexes to LE membranes is dependent on active GTP-Rab7 (Jordens et al., 2001
). Therefore, our findings strongly suggest that interaction of ORP1L with GTP-Rab7 enhances recruitment of dynein/dynactin motor complexes to the endocytic compartments, which leads to enhanced minus-end-directed LE/lysosome movement.
In addition to the LE/lysosome clustering observed in cells expressing ORP1L, transport of the endocytosed marker TRITC-dextran to the late compartments with overexpressed EGFP-ORP1L on their surface was inhibited. Excessive engagement of Rab7 (and associated effectors) in abnormally abundant ORP1L interactions may significantly sequester these components, making them unavailable for their function in the tethering/docking/fusion of multivesicular bodies carrying incoming cargo from early compartments. Furthermore, excess ORP1L on endocytic compartments may sterically hinder normal vesicle interactions. Why EGFP-ORP1L also caused a mild inhibition of TRITC-dextran uptake is unclear. We did not see significant colocalization of the protein with an early endosomal marker, EGFP-Rab5, but there is a significant cytosolic fraction of ORP1L in transfected cells (Johansson et al., 2003
), and this may interfere with components playing a role in early endocytic events.
is a Gram-negative bacterium that inhabits the mucus of human gastric mucosa. This bacterium is a strong risk factor for the development of peptic ulcer disease and adenocarcinoma of the distal stomach (Dunn et al., 1997
). Expression of the virulence factor VacA contributes to the capacity of H. pylori
to colonize the gastric mucosa. A hallmark activity of the VacA protein is the formation of large cytoplasmic vacuoles of LE origin in mammalian cells upon treatment of the cells with weak bases (Papini et al., 2001
; Montecucco and de Bernard, 2003
). This process is inhibited by the dominant negative Rab7 mutant, indicating that it is dependent on the activity of Rab7 (Papini et al., 1997
; Li et al., 2004
). The vacuolation has been suggested to involve formation of anion-selective channels in LE membranes by VacA, resulting in anion influx and stimulation of proton pumping by the vacuolar ATPase. Protonated weak bases such as ammonium chloride accumulate in the endosomes and cause osmotic swelling (Papini et al., 2001
; Li et al., 2004
). We found that full-length ORP1L, but especially the ANK and ANK+PHD fragments, inhibited the vacuolization induced by VacA. The degree of inhibition seemed to correlate with the expression level of the ORP1L construct. Why would binding of ORP1 to GTP-bound Rab7 and the suggested stabilization of the GTP-bound Rab7 on membranes lead to inhibition of the vacuolization, a phenomenon dependent of active Rab7? Again, it is possible that excess ORP1L or the LE clustering induced by ORP1L or its N-terminal fragments renders the endosomal membranes abnormal in functional properties required for VacA-induced cell vacuolation. This could, for example, involve incorporation of the VacA toxin or the function of the vacuolar proton ATPase in the LE membranes. One has to keep in mind that ORP1L may also affect the function of endocytic compartments via Rab7-independent mechanisms.
In summary, the present study identifies ORP1L as a novel interaction partner of Rab7 and demonstrates that overexpression of ORP1L or its truncated fragments modifies the organization and function of late endocytic compartments. This is the first report of a direct connection between the OSBP-related protein family and the master regulators of vesicle transport, the Rab GTPases.