title: RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch.
abstract: Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic.
authors.affiliation: Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Santonico
authors.affiliation: Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Mattioni
authors.affiliation: Department of Cell Biology, University of Calabria, Cosenza, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Panni
authors.affiliation: Department of Clinical and Molecular Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Belleudi
authors.affiliation: Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Mattei
authors.affiliation: 1] Department of Clinical and Molecular Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy [2] Azienda Ospedaliera S. Andrea, Rome, Italy.
authors.forename: M R
authors.initials: MR
authors.lastname: Torrisi
authors.affiliation: 1] Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy [2] IRCCS Fondazione S. Lucia, Rome, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Cesareni
authors.affiliation: Department of Molecular Biology, Tor Vergata University of Rome, Rome, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Castagnoli
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012097:Repressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C407306:GGA adaptor proteins; D018832:Molecular Chaperones; C404508:RNF11 protein, human; D012097:Repressor Proteins; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases