CHX treatment (Fig. S6). The effect of Vps4 on RhoB levels was determined in COS7 cells, which allow high transfection efficiency. As it is shown in Fig. 4d, the decay in RhoB levels upon CHX treatment was slower in Vps4 DN-transfected than in Vps4 wt-transfected cells. Taken together these results indicate that blocking Vps4 function prevents both RhoB sorting into MVB and degradation. The C-Terminal Sequence of RhoB (-CINCCKVL) Acts as a Signal for Protein Endo-Lysosomal Localization and Degradation Lipid modifications of G proteins are key for their specific subcellular localization. To assess the relative importance of the individual lipidic modifications in RhoB localization and stability we generated site-specific mutants. As we reported previously [10], an isoprenylation-deficient mutant, RhoB C193S, failed to associate with cellular membranes and its turnover was completely inhibited (Fig. 5a). Mutation of the first palmitoylation site (C189S), induced a diffuse cytosolic distribution with a perinuclear accumulation and partially impaired degradation, whereas mutation of the second palmitoylation site (C192S) completely stabilized RhoB, same as the double C189,192S mutation. All palmitoylation mutants excluded the nucleus, indicating that isoprenylation is preserved. Thus, both palmitoylation sites contribute to the localization and degradation of RhoB, although to different extents. We next seek to modulate RhoB turnover pharmacologically by interfering with isoprenylation and palmi- toylation. Both simvastatin, an isoprenoid biosynthesis inhibitor, and 2-bromopalmitate, a palmitoylation inhibitor, increased the levels of GFP-RhoB and endogenous RhoB, but not of RhoA (Fig. S7). Moreover, short-term treatment (3 h) with either inhibitor elicited the appearance of diffusely distributed GFP-RhoB, which excluded the nucleus in the case of 2-BP, but not in the case of simvastatin-treated cells (Fig. S7c). Since isoprenylation is irreversible, the effect of simvastatin is consistent with the diffuse localization of the newly synthesized non-processed protein. Thus, isoprenylation and palmitoylation may act as switches for RhoB degradation. RhoA is 86% identical to RhoB differing mainly in the C- terminal sequence. As shown in Fig. 5a, GFP-RhoA was cytosolic and stable, suggesting that RhoB C-terminus contains the structural determinants for degradation. To test this hypothesis, we prepared chimeric proteins of GFP with several short sequences from RhoB C-terminus (Fig. 5b). Interestingly, chimeric proteins containing only the last 22, or even the last 8 amino acids of RhoB, showed a GFP-RhoB-like distribution, and were not stable. In contrast, the fusion protein containing only the CAAX box appeared diffuse with a perinuclear accumulation, and was not detectably degraded. Thus, the isoprenylation motif alone is not sufficient, but a sequence containing the isoprenylation and the two palmitoylation sites of RhoB (CINCCKVL) efficiently directs protein localization and degradation. In order to investigate if this sequence was targeting chimeric proteins for degradation through a lysosomal pathway, we assessed the localization of the GFP-CINCCKVL construct, termed GFP- 8, in the presence of lysosomal inhibitors. GFP-8 significantly co- localized with RFP-RhoB and LTR in control cells (Fig. 6a). Moreover, in cells treated with chloroquine or with bafilomycin A1 GFP-8 was retained inside LTR-positive dilated vesicles, likewise GFP-RhoB/LTR (Fig. 2c). Furthermore, GFP-8 accumulated in U18666A-induced dense MVB visible by DIC (Fig. 6b), where it co-localized with RFP-RhoB and LBPA (Fig. 6c). Thus, GFP-8 is Lysosomal Degradation Sequence internalized into MVB by mechanisms similar to those operating on RhoB. Targeting Stable Proteins for Rapid Lysosomal Degradation through the CINCCKVL Sequence To assess the ability of the CINCCKVL sequence to induce endo-lysosomal localization and rapid degradation of stable cytosolic proteins we created additional chimeras. TagRFP, a monomeric protein only 29% identical to GFP, was diffuse and did not co-localize with GFP-RhoB or Lamp1-GFP (Fig. 7a). In contrast, the chimeric protein tagRFP-CINCCKVL (tagRFP-8) co-localized with GFP-RhoB-positive vesicles and appeared inside Lamp1-GFP-delimited vesicles. In chloroquine-treated cells, tagRFP remained excluded from RhoB-containing and Lamp1- delimited dilated MVB, whereas tagRFP-8 was totally accumu- lated inside. Moreover, whereas tagRFP was stable, tagRFP-8 reached lower levels and was rapidly degraded (estimated half-life of tagRFP-8 #3 h; Fig. 7b). As expected, tagRFP-8 migrated faster than tagRFP in SDS-PAGE gels, a feature typical of other isoprenylated and palmitoylated proteins, like RhoB or H-Ras [10,33]. As shown above, the levels of the cytosolic protein RhoGDI are not altered by agents modulating lysosomal protein degradation or endosomal traffic and they remain stable after inhibition of protein synthesis (Fig. 1). Therefore, we considered it a good candidate to confirm the effect of the addition of the CINCCKVL sequence to its C-terminal end. Whereas GFP-RhoGDI was diffuse, GFP- RhoGDI-CINCCKVL (GFP-RhoGDI-8) was associated with LTR-positive cytosolic vesicles and strongly co-localized with RFP-RhoB (Fig. 7c). Moreover, in chloroquine-treated cells, GFP- RhoGDI was excluded from, whereas GFP-RhoGDI-8 was accumulated in LTR and RhoB-positive dilated vesicles (Fig. S8), thus corroborating the lysosomal localization of the chimeric protein. GFP-RhoGDI-8 lysosomal targeting was sensitive to modulation of isoprenylation. Treatment of cells expressing membrane and vesicle-associated GFP-RhoGDI-8 with simvasta- tin led to its diffuse localization throughout the cytosol (Fig. 7d, a and b). Conversely, when cells were transfected with GFP- RhoGDI-8 in the presence of simvastatin, the protein was completely cytosolic (Fig. 7d c) but removal of the statin allowed its appearance in cytosolic vesicles (Fig. 7d d). As observed with other -CINCCKVL chimeric proteins, the levels and the half-life of GFP-RhoGDI-8 were consistently lower than those of GFP-RhoGDI and endogenous RhoGDI (Fig. 7e). Interestingly, simvastatin increased the levels of GFP-RhoGDI-8 but not of GFP-RhoGDI (Fig. 7f). These results indicate that C- terminal processing can act as a switch for the localization and degradation not only of RhoB but of chimeric proteins bearing the CINCCKVL motif. The specific subcellular localization and reduced protein stability of GFP-RhoGDI-8 (half-life #3 h) were also observed in stably transfected NIH-3T3 fibroblasts (Fig. S8), ruling out a potential interference of lipid-mediated transfection in the effects observed. Finally, we confirmed that the CINCCKVL motif rendered GFP-RhoGDI-8 susceptible to modulation by lysosomal proteolysis inhibitors. Leupeptin and chloroquine increased GFP-RhoGDI-8 levels, whereas GFP- RhoGDI and endogenous RhoGDI remained unchanged (Fig. 7f). Taken together, these results show that the CINCCKVL sequence is capable of promoting a RhoB-like localization and degradation rate when fused to the C-terminal end of unrelated proteins like GFP, tagRFP and GFP-RhoGDI. Moreover, both the levels and distribution of the chimeric proteins can be regulated pharmacologically by means of widely used drugs, such as statins. PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8117