The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo-Lysosomal Pathway Dolores Pe´ rez-Sala1.*, Patricia Boya2., Irene Ramos1, Mo´ nica Herrera1, Konstantinos Stamatakis1 1 Department of Chemical and Physical Biology, Centro de Investigaciones Biol o´gicas, Consejo Superior de Investigaciones Cient´ıficas, Madrid, Spain, 2 Department of Molecular and Cellular Medicine, Centro de Investigaciones Biolo´ gicas, Consejo Superior de Investigaciones Cient´ıficas, Madrid, Spain Abstract Background: Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. Methodology/Principal Findings: By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. Conclusions/Significance: Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs. Citation: Pe´rez-Sala D, Boya P, Ramos I, Herrera M, Stamatakis K (2009) The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo- Lysosomal Pathway. PLoS ONE 4(12): e8117. doi:10.1371/journal.pone.0008117 Editor: Charleen T. Chu, University of Pittsburgh, United States of America Received September 9, 2009; Accepted November 5, 2009; Published December 2, 2009 Copyright: 2009 Pe´rez-Sala et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from MCI, SAF2006-03489 and RETICS ‘‘RIRAAF’’ RD07/0064/0007 from ISCIII to DPS and BFU2006-00508 to PB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dperezsala@cib.csic.es . These authors contributed equally to this work. Introduction The degradation of cellular components plays a key role in the maintenance of cellular functions. Protein degradation serves regulatory roles in cell cycle and signalling and provides amino acid supply for protein synthesis. The main proteolytic systems in the cell are the proteasome machinery and the lysosomal pathway. Whereas the proteasome has been mainly involved in the rapid degradation of regulatory or misfolded proteins [1], lysosomes are the sites where several degradation pathways converge, including endocytic and autophagic pathways [2]. GTPases of the Rho family play crucial roles in the regulation of the actin cytoskeleton, cell survival and gene expression. Among Rho proteins, RhoB displays unique functions in the control of endocytic traffic, influencing the sorting of signalling kinases [3–5] and growth factor receptors [4,6]. This has important consequences for growth factor signalling, cell survival and proliferation, contributing to the proposed role of RhoB in tumor suppression [7,8]. RhoB is also unique among Rho proteins due to its short half life, which has been estimated in 2–3 h [9,10]. RhoB is an immediate early gene which is rapidly induced by DNA damage or growth factors [11,12]. Thus, maintaining a high RhoB turnover is important to ensure the rapid response of RhoB levels to various stimuli. However, the molecular basis for RhoB instability has not been elucidated. From a structural point of view, RhoB is highly homologous to RhoA. However, whereas RhoA is retained in the cytosol bound to RhoGDI under resting conditions [13], RhoB is mainly a membrane-associated protein, even in non-stimulated cells [10], and both plasma membrane and endosomal localizations of RhoB have been reported [14,15]. The structural basis for these differences resides in the hypervariable C-terminal domain of these proteins. Whereas RhoA is geranylgeranylated and possesses polybasic sequence, RhoB presents two palmitoylated cysteine residues (C189 and C192) close to the isoprenylation motif [16]. Hypervariable domains of monomeric GTPases of the Rho and Ras families display unique features including distinct distributions of charged amino acids, phosphorylation sites and lipid moieties [17,18], which mediate specific membrane localization and orientation, traffic and interaction with effectors [19,20]. Howev- er, the role of hypervariable domains of G proteins in protein stability is poorly understood. Here we have explored the cellular pathways involved in RhoB degradation and the structural determinants directing this process. Our results reveal several novel striking features of RhoB: 1) this short-lived protein is degraded through a lysosomal pathway; 2) both, isoprenylation and palmitoylation are required for RhoB degradation; and 3) the last eight amino acids of RhoB, which comprise the posttranslational modification motif, are sufficient to PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8117