from lysosomes, resulting in increased stability [39–42]. The CINCCKVL sequence is unique to RhoB as determined by BLAST and is conserved in all species in which the RhoB sequence is available. However, several proteins belonging to the Ras superfamily, including H-Ras, TC10 and Rap2 possess palmitoylated cysteines close to the isoprenylated C-terminus. Nevertheless, these proteins contain specific features in their hypervariable domains including distinct distributions of acidic and basic amino acids and/or spacing of lipidated residues that result in a different subcellular localization in spite of possessing a lipidation motif similar to that of RhoB [20]. More distantly, in yeast, palmitoylation of Vac8 and Ykt6, the latter being also isoprenylated, has been found to direct localization at the vacuole [43,44], although at present, there is no information on its role on protein stability. Future studies will address whether similar mechanisms may operate for other proteins. Our results show that the sequence containing RhoB isopreny- lation and palmitoylation sites (CINCCKVL) is sufficient to promote a RhoB-like localization and degradation rate when fused to the C-terminal end of several unrelated proteins. Therefore, an interesting aspect of our studies is the potential use of the CINCCKVL sequence to control the localization and cellular levels of proteins of interest. For instance, chimeric proteins bearing this sequence could be potentially used for delivering bioactive components to the endo-lysosomal compartment to modulate lysosomal function, which has broad implications for cell viability, both in carcinogenesis and degenerative processes. Moreover, levels of CICNCKVL-ending proteins can be increased by lysosomal protease inhibitors. This raises the possibility of using these chimeric proteins as reporters of lysosomal proteolytic activity. Importantly, isoprenylation and palmitoylation can act as switches for the lysosomal targeting and degradation of chimeric proteins bearing CINCCKVL C-terminal sequences. Thus, levels of CINCCKVL-ending chimeric proteins can be regulated by commonly used drugs such as simvastatin. Also, given the increasing interest for the clinical use of isoprenylation inhibitors, the potential for use of CINCCKVL contructs for monitoring isoprenylation efficiency in living cells can be hypothesized. Finally, identification of this novel protein degradation sequence may provide valuable tools to study the mechanisms of MVB formation and lysosomal proteolysis in health and disease. Materials and Methods Cell Culture and Treatments BAEC were obtained and cultured as previously described [10]. NIH-3T3 fibroblasts or COS7 cells, cultured as described [10] were treated with the indicated agents or vehicle in the absence of serum. Human primary fibroblasts from apparently healthy subjects and Niemann-Pick disease patients were from Coriell Institute for Medical Research (Candem, NJ). MEFs from wt and Atg5(2/2) mice were kindly provided by Dr. N. Mizushima. Antibodies and Reagents Antibodies for Western blot were anti-RhoB (sc-180), anti- RhoGDI (sc-360), anti-RhoA (sc-418), and anti-ubiquitin (sc- 8017), from Santa Cruz Biotechnology (Heildelberg, Germany), anti-GFP from Clontech (Palo Alto, CA) and anti-tagRFP from Evrogen (Moscow, Russia). Antibodies for IF were anti-RhoB (sc- 8048) and anti-RhoGDI (sc-360), from Santa Cruz Biotechnology, and anti-cathepsin B from Calbiochem. Anti-LBPA was from Echelon Biosciences Inc. (Salt Lake City, UT). Bafilomycin A1 was from A.G. Scientific, Inc. (San Diego, CA). LTR was from Lysosomal Degradation Sequence Molecular Probes (Invitrogen). Simvastatin was a gift form Merck, Sharp & Dohme (Madrid, Spain). Plasmids and Transfections pEGFP-C1 and pDsRed1-C1 were from Clontech. pTagRFP-C was from Evrogen. Human RhoGDI cDNA was from Origene (Rockville, MD). The pcDNA3-HA-RhoB plasmid was generously provided by Dr. G.C. Prendergast. Lamp1-GFP was from Prof. Jennifer Lippincott-Schwartz (NIH, Bethesda, MD). GFP-Rab7 wt was kindly provided by Prof. Cecilia Bucci (University of Copenha- gen, Denmark). GFP-Vps4 wt and E334Q were the generous gift of Dr. Philip Woodman (University of Manchester, UK). The generation of pEGFP-C1-HA-RhoB wt and C193S mutant has been described [10]. Palmitoylation-deficient mutants of RhoB were generated by PCR using oligonucleotides: 59-GCTCTA- GATCATAGCACCTTGCACGAGTTGATGC-39, 59-GCTC- TAGATCATAGCACCTTGCAGCAGTTGATCGAGCCATT- CTG-39, and 59-GCTCTAGATCATAGCACCTTGCACGAG- TTGATCGAGCCATTCTG-39 for the C192S, C189S, and C189,192S mutants, respectively. Mutation sites are underlined. Constructs encoding chimeric proteins comprising GFP and various sequences from the RhoB or TC-10 C-terminus, were generated by PCR using pEGFP-C1 as a template. The forward primer was common for all constructs: 59-GAGTAGAAGCT- TATGGTGAGCAAGGGCGAGGAG-39. Reverse primers were 59-CCGAATTCTAGAGGACCTTGCACTTGTACAGCTCGT- CC-39, 59-CCGAATTCTAGAGGACCTTGCAACAGTTGAT- ACACTTGTACAGCTCGTCCATGC-39 and 59-CCGAATT- CTCACGTAATTAAACAACAGTTGATACACTTGTA CAG- CTCGTCCATGC-39 for generating the GFP-CKVL, GFP-CI- NCCKVL (GFP-8) and GFP-CINCCLIT (GFP-8T) constructs, respectively. PCR products were digested with HindIII and EcoRI and cloned into pcDNA3.0. The construct encoding GFP plus the last 22 amino acids of RhoB was generated by amplifying GFP with a BssHII site at its 39 end, using pEGFP-C1 as a template and primers, forward 59-GAGTAGAAGCTTATGGTGAGCAAGG- GCGAGGAG-39, and reverse 59-CATCTTGCGCGCGTCT- TGTACAGCTCGTCC-39. The PCR product was subcloned into the HindIII-BssHII sites of pcDNA-RhoB. The BssHII site is located at position 806 of the sequence of the rat RhoB gene (NM_022542). Therefore, this construct contains the C-terminal sequence of RhoB from T175. pRFP-RhoB was generated by cloning HA-RhoB into the BglII-EcoRI sites of pRFP. For generation of the construct encoding tagRFP-CINCCKVL, tagRFP was amplified by PCR using pTagRFP-C as template and oligonucleotides, forward 59- CCGCTAGCGCTACCGG- TCGCCACCATGG-39 and reverse, 59- CCGGATCCCTA- GAGGACCTTGCAACAGTTGATACAATTAA GTTTGTGC- CCCAGTTTGC-39. The PCR product was subcloned into the NheI-BamHI sites of the pTagRFP-C plasmid. The cDNA of human RhoGDI (Origene Technologies, Inc. Rockville, MD) was amplified with oligonucleotides introducing restriction sites for SacI or Eco RI: forward primer 59- CCGAGCTCAAATGGCT- GAGCAGGAGCCC-39, reverse primer 59- CCGAATTCT- CAGTCCTTCCAGTCCTTCTTGATGG-39. The PCR prod- uct was cloned into the SacI-EcoRI sites of the pEGFP-C1 plasmid. For generation of GFP-RhoGDI-CINCCKVL, the same strategy was used except that the oligonucleotide 59- CCGAATTCCTAGAGGACCTTGCAACAGTTGATACAG- TCCTTCCAGTCCTTCTTGATGG-39 was used as reverse primer. Confluent BAEC or NIH-3T3 fibroblasts or COS7 cells at 80% confluence were transfected with Lipofectamine 2000 (Invitrogen) using 1 mg of construct following the manufacturer instructions. PLoS ONE | www.plosone.org 11 December 2009 | Volume 4 | Issue 12 | e8117