Intracellular Transport, Assembly, and Degradation of Wild-Type and Disease-linked Mutant Gap Junction Proteins

Abstract

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t1/2 < 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway.

Identification of a COOH-terminal Segment Involved in Maturation and Stability of Human Ether-a-go-go-related Gene Potassium Channels

“…Consistent with this view, we found that Golgi transit of HERG ⌬860 -899 could not be restored by incubating the cells at room temperature for 12 h (not shown). Similar insensitivity to glycerol and temperature have been described for other HERG mutants (36) as well as for distinct membrane proteins and ion channels that exhibit defective trafficking (37,38). Although the basis of transport block of this class of mutant proteins is unknown, it is possible that these mutations cause a major collapse in protein struc-2 A. Akhavan, R. Atanasiu, and A. Shrier, unpublished data.…”

Oculodentodigital Dysplasia-causing Connexin43 Mutants Are Non-functional and Exhibit Dominant Effects on Wild-type Connexin43

“…GFPtagged connexins have been widely reported to be functional with essentially unaltered transport and assembly properties (5, 39 -41). Subpopulations of wild-type and Cx43 mutants localized to lysosomes and the Golgi apparatus were not unexpected given the role of lysosomes in Cx43 degradation (32,42,43) and the relatively short half-life (1.5-3.5 h) of Cx43 (44 -46) requiring continual gap junction formation and turnover. Because patients suffering from ODDD would not be expected to express the mutant in the absence of wild-type Cx43, the sole expression of G21R or G138R in N2A or HeLa cells provides valuable insight into the functional state of the mutant but does not accurately mimic the human disease where the mutant and wild-type forms of the protein are likely expressed in equal amounts.…”

“…In some cases, these could be considered gain-of-function mutations. The third class of mutants could involve altered intracellular transport and assembly as with some Cx32 mutants associated with Charcot-Marie-Tooth disease (32) or Cx26 mutations linked to deafness and skin disease (33)(34)(35). This class of mutants may in fact be functional if transported to the cell surface in association with wild-type proteins, not unlike mutations of the chloride channel responsible for cystic fibrosis (36).…”

A Truncated Isoform of c-Mpl with an Essential C-terminal Peptide Targets the Full-length Receptor for Degradation

“…In both mammalian cells and in yeast, it has been shown that a post-endoplasmic reticulum quality control mechanism appears to be responsible for targeting and lysosomal/vacuolar degradation of misfolded membrane proteins (42)(43)(44)(45). The presence of a cytosolic portion may be a prerequisite for the detection of partly unfolded Mpl by the post-endoplasmic reticulum quality surveillance system.…”