Lysosomal Degradation Sequence Figure 6. Localization of GFP-CINCCKVL in endo-lysosomal compartments. (a) BAEC transfected with RFP-RhoB and/or GFP-CINCCKVL (GFP-8) were treated with vehicle, 10 mM chloroquine or 5 nM bafilomycin A1, incubated with 25 nM LTR when indicated and visualized live by confocal microscopy. (b) BAEC transfected with GFP-8 were treated with 10 mM U18666A for 24 h. DIC and fluorescence images showing dense lysosomes and GFP-8 accumulations are shown. (c) BAEC transfected with GFP-8 and RFP-RhoB and treated with U18666A as above were fixed and co-localization of GFP-8, RFP-RhoB and LBPA was detected by IF. Bars, 20 mm. doi:10.1371/journal.pone.0008117.g006 Discussion Rapid protein degradation contributes to the tight control of RhoB levels. The study of the mechanisms responsible for RhoB degradation points to the existence of a degradation pathway (schematized in Fig. S9) by which RhoB, a cytosolic protein, is delivered into MVB and is degraded in acidic compartments. This process depends on the function of proteins of the Vps machinery and on RhoB posttranslational modification, which mediates its association with late endosomes. Moreover, a sequence of only eight amino acids comprising the RhoB lipidation motif can ‘‘pull’’ other proteins towards an endo-lysosomal compartment for rapid degradation. The finding that the lysosomal pathway achieves the rapid degradation of RhoB is relevant because the proteasome is considered responsible for the degradation of most short-lived proteins [1]. Autophagy constitutes the major pathway leading to lysosomal degradation [23]. Our results indicate that macro- autophagy is not responsible for RhoB degradation. This is consistent with observations indicating that autophagy does not play an obvious part in the degradation of short-lived proteins, even under serum deprivation conditions [34]. On the other hand, the CINCCKVL sequence does not respond to the any of the consensus sequences that elicit chaperone-mediated autophagy [35]. In contrast, our results imply that RhoB reaches lysosomes through the endocytic pathway. RhoB is found in MVB and is strongly accumulated in this compartment when the MVB lipids are selectively altered either by pharmacological means, such as U18666A and endocytosed anti-LPBA antibodies, or by genetic disease. Although these conditions clearly affect MVB membrane dynamics, it is not clear how they would affect lysosomal degradation. Endocytosed anti-LBPA antibodies do not impair EGFR degradation [36], whereas U18666A reduces late endo- some acidification. Thus the lack of a detectable effect of this compound on RhoB degradation could be the net result of increased invagination and compromised proteolytic activity [37]. It is interesting to note that lipid-mediated ‘‘trapping’’ of RhoB into MVB is selective since Rab7, which is also associated with the late endosome limiting membrane, is not accumulated in this compartment. This could arise from the strong binding of RhoB to defined endosomal membrane domains resulting in its remaining associated with invaginating membranes. In fact, RhoB extraction from membranes requires high detergent concentrations (unpub- lished observations). Given the clear role of MVB membrane invagination in RhoB sorting we used a powerful strategy to block this process. DN Vps4 brings about an overall impairment of the ESCRT machinery and blocks intraluminal vesicle formation [38]. The inhibition of RhoB degradation specifically observed in Vps4 DN-transfected cells confirms that RhoB is degraded through an endo-lysosomal pathway requiring MVB biogenesis. The observation that palmitoylation appears to play a positive role in RhoB degradation constitutes a singular case since, in mammalian cells, palmitoylation most often targets proteins away PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8117