Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus. INTRODUCTIONConnexins form gap junction channels that mediate intercellular communication by allowing the direct transfer of ions and small aqueous molecules between neighboring cells or by serving as hemichannels at the plasma membrane (Harris, 2001;Goodenough and Paul, 2003;Saez et al., 2003;Laird, 2006). There are Ͼ20 human connexin genes and one of the most ubiquitously expressed is connexin43 (Cx43). Several diseases have been linked to mutations in different connexin genes, including Cx43 (Kelsell et al., 2001;Laird, 2008). Many of these mutant connexins are not efficiently processed and lack the ability to form functional gap junction channels. Determining how mutant connexins are mistargeted requires a more complete understanding of how normal connexins are processed by the cell.A gap junction hemichannel consists of six connexins, which oligomerize before delivery to the plasma membrane (Martin and Evans, 2004;Segretain and Falk, 2004;Koval, 2006). Unlike most multimeric membrane channels, Cx43 is unusual in that it does not oligomerize in the endoplasmic reticulum (ER) (Musil and Goodenough, 1993), which is more commonly a prerequisite to further transport along the secretory pathway (Ellgaard and Helenius, 2003;Anelli and Sitia, 2008). Instead, Cx43 is transported out of the ER as an apparent monomer that then oligomerizes in the Golgi complex (Musil and Goodenough, 1993;Koval et al., 1997). Although other connexins, such as Cx46 (Koval et al., 1997), also oligomerize in the Golgi apparatus, this is not a universal pathway for connexin oligomerization because other gap junction proteins, such as Cx32, preferentially oligomerize in the ER (Martin and Evans, 2004;Koval, 2006). Understanding the molecular basis for this difference, as well as the ability of monomeric Cx43 to be transported from the ER to the Golgi apparatus has proven difficult, because little is known about chaperones that regulate connexin folding. In particular, it seemed likely that one or more chaperones would be requir...

Connexin oligomerization and trafficking are regulated processes. To identify proteins that control connexin43 (Cx43), a screen was designed using HeLa cells expressing a Cx43 construct with di-lysine endoplasmic reticulum (ER)-retention/retrieval motif, Cx43-HKKSL. At moderate levels of expression, Cx43-HKKSL is retained in the ER as monomers; however, Cx43-HKKSL stably overexpressed by HeLa cells localizes to the perinuclear region and oligomerizes. HeLa/Cx43-HKKSL overexpressors were transiently transfected with pooled clones from a human kidney cDNA library and used immunofluorescence microscopy to identify cDNAs that enabled overexpressed Cx43-HKKSL to convert from a perinuclear to ER localization pattern. Using this approach, a small molecular weight GTPase, rab20, was identified as a candidate protein with the ability to regulate Cx43 trafficking. Enhanced green florescent protein (EGFP)-tagged rab20 showed a predominantly perinuclear and ER localization pattern and caused wild-type Cx43 to be retained inside the cell. By contrast, mutant EGFP-rab20T19N, which lacks the ability to bind GTP, had no effect on Cx43. These results suggest Cx43 is transported through an intracellular compartment regulated by rab20 along the secretory pathway.