ERp29 Restricts Connexin43 Oligomerization in the Endoplasmic Reticulum

Abstract

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...

Structural Determinants and Proliferative Consequences of Connexin 37 Hemichannel Function in Insulinoma Cells

“…Less clear is whether these disulfide bonds are required for HC function once oligomerized as reconstituted Cx26 HCs are functional in the presence of 10 mM DTT (31), which might be expected to break those bonds. For Cx43, ECL stability in the endoplasmic reticulum is conferred by association with chaperone proteins that guide Cx monomers to the trans-Golgi network (32), where disulfide bonds between ECL cysteines form and oligomerization occurs (30). However, disulfide bond formation in Cx43 is not necessary for trafficking to the plasma membrane nor HC function, only for HC docking to form GJCs (14,15).…”

“…Cx37 appears to display properties of both these Cxs. Like Cx26, Cx37 oligomerizes in the endoplasmic reticulum (33), but unlike Cx26, disulfide bond formation between ECL cysteines is not necessary for oligomerization (14,15); if chaperone proteins interact with Cx37 ECLs, they clearly do not interfere with oligomerization in the endoplasmic reticulum, like they do for Cx43 (32). In contrast, like Cx43, Cx37 traffics to the cell surface with and without disulfide bond formation between ECL cysteines; however, unlike Cx43, Cx37 without ECL cysteines is unable to form obviously functional HCs.…”

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

“…Other than Cx46, which has been shown to have a slow turnover rate in enucleated lens fibre cells (Jiang et al, 1993), connexins are presumed to have similar life cycles to that of Cx43 (also known as GJA1) (Laird, 2006). Generally, connexins are co-translationally inserted into endoplasmic reticulum (ER) membranes before trafficking as monomers or oligomers (Das et al, 2009) to the trans-Golgi network where they complete their oligomerization into connexons (Musil and Goodenough, 1993;Vanslyke et al, 2009). Vesicular and tubular post-Golgi carriers transport connexons to the cell surface in a process that, for Cx43 at least, requires intact microtubules (Thomas et al, 2001;Thomas et al, 2005;Shaw et al, 2007).…”

Characterization of ERp29 and ADP-Ribosylation Factor 5 Interaction

“…세포 내에서 ERp29는 단백질 의 분비를 촉진시키며, 정자형성과정과 방사선 조사, 손상된 척수에서 특이적으로 발현한다 [4,10]. 한편 ERp29가 connexin43 oligomerization과 apoptosis를 저해하고, 반대로 cytokeratin 19는 ERp29표현을 저해한다 [1,2] …”