The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia.

Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome-the trafficking complex in charge of retrieving GPCRs from cilia-engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia.

Regulated trafficking of G protein-coupled receptors (GPCRs) controls cilium-based signaling pathways. ß-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between ß-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a ß-arrestin-dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

Post translational modifications of Rab GTPases.

Rab GTPases, the highly conserved members of Ras GTPase superfamily are central players in the vesicular trafficking. They are critically involved in intracellular trafficking pathway, beginning from formation of vesicles on donor membranes, defining trafficking specificity to facilitating vesicle docking on target membranes. Given the dynamic roles of Rabs during different stages of vesicular trafficking, mechanisms for their spatial and temporal regulation are crucial for normal cellular function. Regulation of Rab GTPase activity, localization and function has always been focused in and around the association of GDP dissociation inhibitor (GDI), Guanine nucleotide Exchange Factor (GEFs) and GTPase accelerating protein (GAP) to Rabs. However, several recent studies have highlighted the importance of different post-translational modifications in regulation of Rab activation and function. This review provides a summary of various post translational modifications (PTMs) and their significance to regulate localization and function of different Rabs.

PTEN Regulates Glucose Transporter Recycling by Impairing SNX27 Retromer Assembly.

The tumor suppressor PTEN executes cellular functions predominantly through its phosphatase activity. Here we identified a phosphatase-independent role for PTEN during vesicular trafficking of the glucose transporter GLUT1. PTEN physically interacts with SNX27, a component of the retromer complex that recycles transmembrane receptors such as GLUT1 from endosomes to the plasma membrane. PTEN binding with SNX27 prevents GLUT1 accumulation at the plasma membrane because of defective recycling and thus reduces cellular glucose uptake. Mechanistically, PTEN blocks the association of SNX27 with VPS26 and thereby hinders assembly of a functional retromer complex during the receptor recycling process. Importantly, we found a PTEN somatic mutation (T401I) that is defective in disrupting the association between SNX27 and VPS26, suggesting a critical role for PTEN in controlling optimal GLUT1 levels at the membrane to prevent tumor progression. Together, our results reveal a fundamental role of PTEN in the regulation of the SNX27 retromer pathway, which governs glucose transport and might contribute to PTEN tumor suppressor function.

A modification switch on a molecular switch: Phosphoregulation of Rab7 during endosome maturation.

Rab GTPases, the highly conserved members of Ras GTPase superfamily are the pivotal regulators of vesicle-mediated trafficking. Rab GTPases, each with a specific subcellular localization, exert tremendous control over various aspects of vesicular transport, identity and dynamics. Several lines of research have established that GDI, GEFs and GAPs are the critical players to orchestrate Rab GTPase activity and function. The importance of post translational modifications in Rab GTPase functional regulation is poorly or not yet been addressed except for prenylation. Our recent study has revealed a novel dephosphorylation dependent regulatory mechanism for Rab7 activity and function. We have shown the importance of PTEN mediated dephosphorylation of Rab7 on highly conserved S72 and Y183 residues, which is essential for its GDI mediated membrane targeting and further activation by GEF. In conclusion, our study highlighted the importance of a phosphorylation/dephosphorylation switch in controlling timely Rab7 localization and activity on endosomes.

PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7.

Tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase that negatively regulates growth factor-induced survival signalling. Here, we demonstrate that PTEN attenuates epidermal growth factor receptor (EGFR) signalling by promoting late endosome maturation by virtue of its protein phosphatase activity. Loss of PTEN impairs the transition of ligand-bound EGFR from early to late endosomes. We unveil Rab7, a critical GTPase for endosome maturation, as a functional PTEN interacting partner. PTEN dephosphorylates Rab7 on two conserved residues S72 and Y183, which are necessary for GDP dissociation inhibitor (GDI)-dependent recruitment of Rab7 on to late endosomes and subsequent maturation. Thus, our findings reveal PTEN-dependent endosome maturation through phosphoregulation of Rab7 as an important route of controlling EGFR signalling.