Taken together, our results Axitinib VEGFR/PDGFR inhibitor indicate that interaction with endosomal lipids is an important determinant in GFP-8 sorting. In all species studied, palmitoylation appears as a key element for lysosomal localization of CINCCKVL chimeras. Protein palmitoylation has been involved in protein sorting, although its effects appear to be cell-type and protein-specific. The mannose receptor N-terminus is palmitoylated and blocking this modification induces lysosomal accumulation of this protein. In the case of the protease-activated receptor 1, palmitoylation allows the correct utilization of tyrosine-based sorting signals, and a palmitoylation-deficient mutant, shows increased degradation in lysosomes. In contrast, the Ca2+ sensor synaptotagmin 7 has been reported to be targeted to lysosomes by its palmitoylationdependent association with the tetraspanin CD63, although the examples of palmitoylation-driven endolysosomal association in mammalian cells are very scarce. In yeast, the palmitoylated protein Vac8 is targeted to the vacuolar membrane, although there is no evidence for vacuolar sorting. Therefore, in most cases, palmitoylation directs proteins away from lysosomes, with only a few examples of palmitoylation-supported ILV sorting. Interestingly, as previously suggested, palmitoylation is also likely responsible for the lack of interaction of GFP-RhoB or GFP-8 with RhoGDI. Nevertheless, it would be interesting to assess whether non-palmitoylated constructs interact with RhoGDI in the cytosol. The human RhoB sequence is not conserved in the lower species, in which its closest homologs are not endosomal proteins. Yet, CINCCKVL chimeric proteins show endolysosomal localization analogous to that found in mammalian cells. In insects, a clear homolog of RhoB has not been identified. However, several proteins exist that possess sequences for bipalmitoylation, although their potential lysosomal targeting has not been explored. In Schizosaccharomyces pombe, the RhoB homolog Rho2 has been reported to be mainly membrane bound and localize at the growing end of the cell and the septation site. Rho2 is isoprenylated and palmitoylated, although, as predicted from its CAAX box sequence, is farnesylated, whereas RhoB is mainly geranylgeranylated in cells. However, when using constructs directing either farnesylation of geranylgeranylation of GFP-8, namely, GFP-CINCCKVL and GFP-CINCCLVM, we did not find significant differences in their lysosomal localization, thus indicating that the length of the isoprenoid moiety is not a critical factor. In addition, Rho2 only possesses one palmitoylation cysteine adjacent to the isoprenylation site. Also in yeast, there are examples of bipalmitoylated proteins, like RasA, which localizes at the plasma membrane, but appears in internal patches when palmitoylation is blocked. However, in this case, the spacing of palmitates is different from that of CINCCKVL proteins.