ternalization [27, 28] by modulating Rac1 activity [28], which controls actin polymerization [29]. In a recent study, we discovered that EHD2 has a dual cellular part and can also serve as a co-repressor of transcription. Entry of EHD2 in to the nucleus is determined by a nuclear localization sequence (NLS) present in its helical domain. We also showed that its exit from the nucleus depends mainly on its SUMOylation (SUMO-small ubiquitin like modifier) [30]. SUMO is often a little molecule (~11 kDa), resembling ubiquitin in its three-dimensional structure [31, 32]. It covalently attaches to target proteins [33] via the acceptor internet site, KxE (in which is definitely an aliphatic branched amino acid and x is any amino acid) [34, 35]. The enzymatic cycle of SUMOylation is similar towards the ubiquitylation cycle [31, 36]. All SUMO proteins are expressed in an immature pro-form, in which they contain a C-terminal stretch of variable length (21 amino acids) just after an invariant Gly-Gly motif that marks the C terminus in the mature protein [37]. Removal of this C-terminal extension by SUMO-specific proteases and exposing the Gly-Gly motif is usually a prerequisite for the conjugation of SUMO to its targets [368]. A wide variety of proteins has been documented to undergo SUMOylation, which affects their stability, localization or activity [39, 40]. At the molecular level, this posttranslational modification modifications the surface of a target protein, enabling/disabling interactions with other proteins [32]. Although numerous endocytic proteins have already been shown to undergo SUMOylation, EHD2 will be the only EHD member shown to be modified by SUMOylation [30]. In the present study we show that EHD3 undergoes Lys315 and Lys511 SUMOylation. We also 10205015 show that SUMOylation of EHD3 is vital for its localization towards the tubular structures of the ERC. Non-SUMOylated EHD3 is concentrated within the perinuclear location with the ERC and delays transferrin recycling, strongly implicating that SUMOylation of EHD3 is vital for tubulation of your ERC and efficient recycling.
HEK293T (human epithelial embryonic kidney cells transformed with 
SV40) (ATCC no. CRL-3216) and COS-7 (CRL-1651) cells had been grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, CA, USA), supplemented with 10% FCS (Beit-Haemek, Israel). All cells have been grown at 37 inside the presence of 5% CO2.
Key antibodies employed were as follows: Mouse monoclonal anti-myc antibody [1:1000 for western blotting (WB), 1:600 for immunoprecipitation (IP), 1:200 for immunofluorescence (IF), Cell Signaling Technologies, Inc. Denver, MA, USA, #2276]; Polyclonal rabbit anti-GFP antibodies (1:1000 for WB, Santa Cruz Biotechnology, Dallas, TX, USA, #sc-8334); Polyclonal rabbit anti-Rab11 (1:30 for IF, Invitrogen, Camarillo, CA, USA, #71300); Monoclonal mouse anti-EEA1 antibody (1:30 for IF, BD Biosciences, 670220-88-9 San-Jose, CA, USA, #610456); Monoclonal Mouse anti-HA antibody (1:1000 for WB, Santa Cruz Biotechnology, Denver, TX, USA, #sc-805). Secondary antibodies incorporated: Peroxide-conjugated goat anti-mouse (1:5000 for WB, #115-035-003); Peroxide-conjugated goat anti-rabbit (1:10,000 for WB, #111-035-144); Cy3-conjugated goat anti-mouse (1:200 for IF, #115-166-072); Rhodamine Red-conjugated goat anti-rabbit (1:200 for IF, #111-295-144). All secondary antibodies have been from Jackson Immunoresearch Laboratories, West Grove, PA, USA).
HAUMO1 was a present from Prof. Michael Nevels (Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany). pEGFPEH