After 24 h cells were lysed and immunoprecipitated with indicated antibodies

After 24 h cells were lysed and immunoprecipitated with indicated antibodies. a complex quality control system that clears diverse proteins independent of sequence or functional similarity (1C3). The unfolded protein response reduces the burden caused by unfolded protein accumulation in the endoplasmic reticulum (ER)2 (4), in part by activating its key regulatory proteins IRE1-XBP1 and ATF6, which results in transcriptional activation of genes important for unfolded protein response, including components of the ER-associated degradation system (ERAD) (5). ERAD is regulated by an ER quality control system that marks proteins that cannot fold or assemble into multiprotein complexes for ubiquitin-dependent degradation (1C3). This system consists of molecular chaperones such as BiP (1C3), which interacts with misfolded protein to enable their transfer across the ER membrane via the multispanning membrane proteins Derlin-1/2/3 and Sec61(6), and the AAA (ATPase p97 (also known as VCP or cdc48)). Subsequent to translocation, misfolded proteins are ubiquitinated via ER-anchored ubiquitin ligases, such as the vertebrate gp78, Parkin, RNF5/RMA1, and Hrd1 (7C10), followed by proteasome-mediated degradation. RNF5 (RING finger domain E3 ligase; also known as RMA1) is one of the few ER-associated E3 Mouse monoclonal to R-spondin1 ubiquitin ligases. Anchored to the ER membrane via its C terminus, RNF5 consists of a classic RING domain (which confers ligase activity), a single transmembrane-spanning domain located within the C-terminal region, and a formin-like homology domain. RNF5 has been shown to promote degradation of misfolded proteins, such as mutant CFTR (CFTR508) (9, 10). Intriguingly, RNF5-mediated degradation of mutant CFTR requires cooperation with another ligase, as has also been shown for gp78 or CHIP, suggesting a two-stage process in which the first ligase promotes substrate mono-ubiquitination, whereas the second mediates poly-ubiquitination (9, 10). Interestingly, independent of a role in recognition and ubiquitination of misfolded proteins, RNF5 has been shown to affect both the stability and localization of cytoskeletal proteins in worms and mammals. Studies from our lab identified RNF5 as a ubiquitin ligase targeting the LIM domain protein UNC-95 for degradation (11). In the muscle, UNC-95 is important for the assembly of dense bodies and M-lines attachment structures that anchor actin-containing thin filaments and myosin-containing thick filaments, respectively, to the muscle cell membrane. The cDNA was amplified by PCR and cloned into BamHI/XhoI sites of pcDNA-FLAG and pEF-hemagglutinin (HA). HA-TCR and p97 constructs were gifts from Dr. R. Kopito (21). FLAG-Rpt4 and -Rpt5 constructs were gifts from Dr. Keiji Tanaka. FLAG- or Myc-tagged full-length human RNF5 was constructed in pEF-FLAG vector or myc-pCDNA3 vectors, respectively. RNF5 was deleted of its C-terminal domain via PCR-based cloning into pEF-FLAG of the DNA fragment corresponding to amino acids 1C164. A RING domain deletion mutant of human RNF5 Nitrofurantoin (from 49 to 268 bp) was generated via PCR-based cloning. CFTR constructs were kindly provided by Dr. Gergely Lukacs. HA-tagged, FLAG-tagged Ubc13 WT or dominant negative constructs (C87A), HA-tagged WT ubiquitin, and HA-tagged ubiquitin mutants (K48R, K63R, K48R/K63R, KO48, and KO63) were generated as described (22, 23). schematic representation of RNF5 constructs. RING mutant RNF5 was generated Nitrofurantoin by deletion of a Nitrofurantoin region encoding 78 amino acids of the RING finger domain. C-terminally truncated RNF5 was created by deleting a region encoding the last 16 amino acids of the protein that contains the transmembrane (JAMP interacts with endogenous RNF5. JAMP was immunoprecipitated (interaction of JAMP with RNF5 requires membrane anchoring. Either the full-length protein, the RING finger deletion mutant, or a C-terminally truncated, Myc-tagged RNF5 were cotransfected with FLAG-JAMP into 293T cells. Proteins were prepared as in and immunoprecipitated with FLAG antibodies followed by immunoblot analysis with Myc antibody or FLAG antibodies as noted in the Western blots shown. JAMP colocalizes with full-length RNF5 but not C-terminally truncated protein. HA-JAMP and FLAG-tagged, full-length RNF5 or C-terminally truncated RNF5 were cotransfected into HeLa cells. Cells were fixed and immunostained with HA and FLAG antibodies after 24 h. of paxillin (13)), we asked whether RNF5 altered JAMP function.