The expression of CIP75 in mouse tissues. A, a DIG-labeled RNA probe to the 3′-UTR of CIP75 was used to probe a commercial membrane containing 2 μg/lane of poly(A) RNA isolated from mouse tissues. A 3.5-kb CIP75 mRNA is expressed in all tissues, to varying degrees. B, a commercial membrane containing 50 μg of protein lysates isolated from mouse tissues was immunoblotted (IB) for CIP75. The CIP75 protein was expressed in all tissues, to varying degrees.

CIP75 interacts with monoubiquitin and Lys⁴⁸-linked tetraubiquitin chain in vitro. A and B, pull-down of His-tagged wild-type CIP75 (CIP75wt), CIP75 deleted for the N-terminal UbL domain (CIP75ΔUbL), and CIP75 deleted for the C-terminal UBA domain (CIP75ΔUBA), with beads alone as a negative control. Interaction of CIP75 and the deletion mutants with GST-tagged monoubiquitin (A) and untagged tetraubiquitin (B) were examined. CIP75wt and CIP75ΔUbL were able to interact with both mono- (A, lanes 2 and 3) and tetraubiquitin (Tetra-UB) (B, lanes 2 and 3), whereas CIP75ΔUBA was not (A and B, lane 4). IB, immunoblot.

CIP75 interacts with non-ubiquitinated Cx43. Sequential immunoprecipitation of ubiquitinated protein and Cx43HKKSL that interacts with CIP75 in lactacystin-treated HeLa-Cx43HKKSL cells. CIP75 was first immunoprecipitated from cellular lysates. Immune complexes were subsequently disrupted using RIPA buffer and subjected to a second immunoprecipitation of Cx43 or HA-tagged ubiquitinated proteins to isolate the subset of proteins that interact with CIP75. CIP75 interacts with Cx43HKKSL and HA-ubiquitin-tagged proteins (top and middle, lane 1). Cx43HKKSL that was initially co-immunoprecipitated with CIP75 is not ubiquitinated (top and middle, lanes 2 and 3), whereas ubiquitinated proteins that were initially co-immunoprecipitated with CIP75 are separately recovered (top, lane 3). CIP75 is not recovered after the sequential immunoprecipitation (bottom, lanes 2 and 3). *, IgG heavy chain; **, light chain. Migration positions of the molecular mass markers in kilodaltons are indicated at the right of the co-immunoprecipitation panels. Protein inputs are shown in the right panels and represent 2% of the amount of lysates used for immunoprecipitations. IP, immunoprecipitation; IB, immunoblot.

CIP75 co-localizes with wild-type and mutant Cx43. HeLa cells were transiently co-transfected with FLAG-CIP75 and ER-retained Cx43 (wild-type (A and E) or lysine mutants (B–D and F)). The subcellular localization of CIP75 (green) and Cx43 (red) with the ER marker calnexin (blue) was visualized by LSCM. CIP75 co-localizes with Cx43, regardless of the extent of lysine to arginine mutations. CIP75 and Cx43 co-localization occurs in an ER-like pattern. Scale bar, 10 μm.

CIP75 interacts with ER-localized Cx43. A, co-immunoprecipitation of CIP75 and Cx43 in HeLa and HeLa-Cx43HKKSL cells. Immunoprecipitated CIP75 interacts with ER-retained Cx43 (A, lane 2), which is not seen in cells that do not express Cx43 (A, lane 1). Immunoprecipitated Cx43 containing an ER retention motif (HKKSL) is able to interact with CIP75 (A, lane 3). The presence of Cx43 does not affect CIP75 expression. Input levels represent 2% of the amount of lysates used for immunoprecipitations. Migration positions of the molecular mass markers in kilodaltons are indicated at the right of the co-immunoprecipitation panels. *, IgG heavy chain. B, HeLa-Cx43HKKSL cells were transiently transfected with FLAG-CIP75. The subcellular localization of CIP75 (green) and Cx43HKKSL (red) with the ER marker calnexin (blue) was visualized by LSCM. Cx43HKKSL localizes to the ER, together with the ER marker calnexin, and CIP75 co-localizes with Cx43HKKSL. Scale bar, 10 μm. IP, immunoprecipitation; IB, immunoblot.

CIP75 interacts with ubiquitinated proteins. A, immunoprecipitation of p27 and CIP75 in untreated or lactacystin-treated HeLa-Cx43HKKSL cells. Lactacystin treatment increases the levels of immunoprecipitated ubiquitinated proteins (A, top, lanes 1 and 3 compared with lanes 2 and 4). CIP75 is not affected by lactacystin treatment (A, bottom, lanes 3 and 4). B, immunoprecipitation of CIP75 in untreated HeLa or HeLa-Cx43HKKSL cells and HeLa-Cx43HKKSL cells that were treated with NH₄Cl, lactacystin, or bortezomib. Immunoprecipitated CIP75 is able to interact with ubiquitinated proteins regardless of Cx43 expression (B, top, lanes 1–5). Treatment with proteasomal inhibitors increases this interaction (B, top, lanes 4 and 5) compared with untreated cells or treatment with a lysosomal inhibitor (B, top, lanes 2 and 3). CIP75 interaction with Cx43 is also increased in cells treated with the proteasomal inhibitors (B, middle, lanes 4 and 5), but CIP75 is not affected by the various treatments (B, bottom, lanes 2–5). Input levels represent 2% of the amount of lysates used for immunoprecipitations. Migration positions of the molecular mass markers in kilodaltons are indicated at the right of the co-immunoprecipitation panels. The amount of proteins in the input material is shown in the right panels of A and B. C, Cx43 levels found in the co-immunoprecipitation assays were quantified with ImageJ and analyzed relative to the immunoprecipitated CIP75 levels with GraphPad Prism 5.0 and SigmaPlot 9.0 software. The data represent the mean of at least four independent experiments ± S.E. (n = 10 for untreated cells, n = 4 for NH₄Cl-treated cells, n = 6 for lactacystin-treated cells, n = 8 for bortezomib-treated cells). **, -fold change in Cx43 levels relative to CIP75 with statistically significant difference (p < 0.01) compared with the untreated control cells. IP, immunoprecipitation; IB, immunoblot.

CIP75 interacts with Cx43 that lacks lysine ubiquitination sites. A, site-directed mutagenesis was performed to mutate lysine residues to arginine to eliminate potential ubiquitination sites. Mutant 1 consists of mutations K264R, K287R, and K303R (red) because these wild-type residues appear to be involved or near those that are involved in CIP75 binding. Mutant 2 consists of the same mutations in Mutant 1 plus the additional mutations K236R, K239R, K243R, K345R, and K346R (green). Mutant 2 represents mutations of all lysine residues in the C-terminal tail. In Mutant 3, all lysine residues in Cx43 are mutated (blue). B and C, co-immunoprecipitation of transiently transfected HeLa cells. B, HeLa cells were transfected with either Cx43HKKSL, Mutant 1, Mutant 2, or Mutant 3. Untransfected HeLa cells were used as the negative control. Immunoprecipitated CIP75 interacted with Cx43HKKSL (B, lane 2), Mutant 1 (B, lane 3), Mutant 2 (B, lane 4), and Mutant 3 (B, lane 5), suggesting that ubiquitination of ER-localized Cx43 is not necessary for the interaction with CIP75. B, HeLa cells were transfected with either Cx43HKKSL, Cx43RRRRISLS, Cx43HKKSL Mutant 3, or Cx43RRRRISLS Mutant 3. CIP75 can also interact with the ER-retained wild-type Cx43RRRRISLS (C, lane 3) and Cx43RRRRISLS Mutant 3 (C, lane 5), which does not contain any lysines for ubiquitination. Input levels represent 2% of the amount of lysates used for immunoprecipitations. IP, immunoprecipitation; IB, immunoblot.

Model of CIP75 facilitating the proteasomal degradation of Cx43. CIP75 may act a shuttle to transport non-ubiquitinated Cx43 from the ER to the proteasome for degradation. CIP75 would bind to the C-terminal tail of ER-localized Cx43 via the UBA domain, perhaps aid in translocating Cx43 out of the ER membrane into the cytosol, and then transport Cx43 to the proteasome, where CIP75 binds to the 19 S proteasome cap subunits Rpn1 and Rpn10 via the CIP75 UbL domain, and Cx43 is subsequently degraded.