We took advantage of this feature to address the key issue of whether a non-lipidated, cytosolic pool of CINCCKVL protein can undergo lipidation to be sorted to the aforementioned membranes or whether sorting must occur on newly synthesized proteins. Using BAEC, which present low cytotoxicity upon treatment with simvastatin, and Dendra2, a protein capable of photoswitching from green to red fluorescence emission after UV light exposure, we set up a simvastatin-based approach to follow specific pools of a Dendra2-CINCCKVL fusion protein. Treatment with simvastatin blocked processing of Dendra-8 rendering a pool of fully cytosolic green construct. This pool was photoswitched obtaining a soluble, non-isoprenylated red fluorescent CINCCKVL construct to follow. Releasing the isoprenylation inhibition by removal of simvastatin allowed endolysosomal targeting of the previously diffuse red construct, clearly demonstrating that Nutlin-3 preexisting CINCCKVL-chimeric proteins may undergo sorting from a cytosolic localization, depending on isoprenoid availability. The observed behavior establishes this sequence as a tunable targeting motif in live cells. The combination of several lipidic modifications, like isoprenylation and palmitoylation constitute important determinants for protein localization in specific subcellular compartments. The spacing of the lipid moieties and the nature of the non-lipidated amino acids may contribute to the generation of unique structures with affinity for membrane domains or protein partners. Here we have shown that the isoprenylation and palmitoylation motif of the GTPase RhoB constitutes one such structure per se and is able to determine protein sorting to lysosomal compartments in cells from distant species, including fungi and humans. Several mechanisms for sorting to MVBs have been reported. The classical mechanism implies ubiquitination of the cargo protein and association with components of the ESCRT machinery, which, according to some models may assemble sequentially to define invagination domains leading to the formation and ultimately the release of ILV. Although under our conditions we have not detected ubiquitination of GFP8, this mechanism cannot be excluded since ubiquitination-independent sorting of certain cargo by ESCRT-mediated processes has also been reported. Recently, a late-endosome microautophagy-like process has been described, which depends on ESCRT I and III for vesicle formation and on Hsc70 for cargo selection, and may mediate the delivery into late endosomes of various cytosolic proteins. Nevertheless, this process does not seem to be involved in sorting of CINCCKVL chimeric proteins since it is disrupted by U18666A, which as shown above, causes strong accumulation of GFP-8 in MVB. In turn, although the autophagic and endolysosomal pathways may converge at several levels, we have previously observed that GFP-8 and the autophagosomal marker RFP-LC3 show different distribution patterns.