Lysosomal Degradation Sequence direct the endo-lysosomal degradation of chimeric proteins. Taken together, our observations delineate a novel pathway for protein degradation. Results RhoB Is Degraded through a Lysosomal Pathway Protein degradation is crucial for cell homeostasis. The main pathways for protein degradation are the ubiquitin proteasome system and the lysosomal pathway. To elucidate the involvement of these pathways in RhoB turnover, we used a battery of protease inhibitors with different specificity towards both pathways (Fig. 1a). The highly selective proteasome inhibitor lactacystin, did not increase RhoB, whereas Z-LLL-CHO, which is less specific [21], moderately increased RhoB levels. ALLM, a calpain-2 inhibitor, and leupeptin, a thiol and serine protease inhibitor clearly increased RhoB. Interestingly, the most effective compound was chloroquine, an inhibitor of lysosomal acidification. Ubiquitinated protein levels, indicative of proteasome inhibition, did not correlate with increases in RhoB. None of the inhibitors increased the levels of RhoGDI, which was used as control (Fig. 1a). Moreover, inhibition of lysosomal degradation stabilized RhoB. RhoB protein levels rapidly decayed in control cells upon inhibition of protein synthesis with cycloheximide (CHX) (Fig. 1b). However, pre-treatment with thiol protease inhibitors, leupeptin plus E64d, or with chloroquine stabilized RhoB, suggesting that RhoB is rapidly degraded by lysosomal proteases. Interestingly, RhoB stabilization was also observed after disrupting Figure 1. RhoB is degraded through a lysosomal pathway. (a) Confluent serum-deprived bovine aortic endothelial cells (BAEC) were treated with 10 mM lactacystin, 50 nM Z-LLL-CHO, 10 mM ALLM, 50 mg/ml leupeptin or 10 mM chloroquine for 24 h. Cell lysates (20 mg of protein) were analyzed by Western blot. RhoB and ubiquitinated protein levels corrected by RhoGDI levels are shown as average values 6 SEM of three experiments (*p,0.05 vs control). (b) BAEC were pre-treated with the inhibitors for 30 min. NH4Cl and bafilomycin A1 were added at 30 mM and 5 nM, final concentrations, respectively. RhoB levels were assessed before and after treatment with CHX (20 mg/ml). Levels of RhoGDI were used as control. The percentage of RhoB remaining after 3 h CHX treatment is shown as average values 6 SEM of at least three different experiments (*p,0.05 vs control). (c) BAEC were treated with 50 mg/ml leupeptin plus 5 mg/ml E64d or 10 mM chloroquine for 24 h and visualized by immunofluorescence (IF) of endogenous RhoB (green) and staining with 500 nM LTR (red) and DAPI (blue). Images shown are single confocal sections. Bar, 20 mm. (d) BAEC were treated with 30 mM NH4Cl or 5 nM bafilomycin A1 for 24 h and fixed. Nuclei were stained with DAPI (blue) and RhoB (green) was detected by IF and confocal microscopy. Images are shown as maximal projections of confocal planes of 0.5 mm Z-sections. Bar, 20 mm. (e) BAEC subjected to osmotic shock for 5 min were allowed to recover in the absence or presence of 10 mM potassium chloride (K+) for 3 h, during which CHX (20 mg/ml) was present to block new protein synthesis. RhoB levels were assessed by Western blot. doi:10.1371/journal.pone.0008117.g001 PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8117