Dislocation and degradation from the ER are regulated by cytosolic stress

Akey step in ER-associated degradation (ERAD) is dislocation of the substrate protein from the ER into the cytosol to gain access to the proteasome. Very little is known about how this process is regulated, especially in the case of polytopic proteins. Using pulse-chase analysis combined with subcellular fractionation, we show that connexins, the four transmembrane structural components of gap junctions, can be chased in an intact form from the ER membrane into the cytosol of proteasome inhibitor–treated cells. Dislocation of endogenously expressed connexin from the ER was reduced 50–80% when the cytosolic heat shock response was induced by mild oxidative or thermal stress, but not by treatments that instead upregulate the ER unfolded protein response. Cytosolic but not ER stresses slowed the normally rapid degradation of connexins, and led to a striking increase in gap junction formation and function in otherwise assembly-inefficient cell types. These treatments also inhibited the dislocation and turnover of a connexin-unrelated ERAD substrate, unassembled major histocompatibility complex class I heavy chain. Our findings demonstrate that dislocation is negatively regulated by physiologically relevant, nonlethal stress. They also reveal a previously unrecognized relationship between cytosolic stress and intercellular communication

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

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 \u3c 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

Proteolytic maturation of vaccinia virus structural proteins

Vaccinia virus (VV) is a large DNA virus belonging to the Orthopoxvirus family. The viral replicative life cycle takes place solely within the cytoplasm of a mammalian host cell. The VV genome contains 196 open reading frames which are expressed in a highly regulated and temporal fashion in order to bring about the production of a mature virion. In the process of viral replication many VV proteins are synthesized that require posttranslational modifications to become functional. A few of these modifications include, glycosylation, ADP-ribosylation, phosphorylation, fatty acid acylation, and proteolytic processing. This last modification is especially important with regard to the structural proteins of the virus in that they undergo prysis for an infectious virus particle to be formed, a common theme in viral systems. In order to understand these events in more detail, three abundant virion protein constituents 4a, 4b, and 25K were chosen as models for study. The three main questions we wanted to answer were: Is there a cleavage consensus site within the precursors, what protease(s) and/or factors are necessary for the process, and how are the events regulated in vivo? Our approach included development of specific immunological reagents to identify cleavage products as well as to show where these core proteins are located during virion assembly. We have subsequently identified cleavage products by N-terminal microsequence from each of the three structural proteins and this information has elucidated a putative cleavage consensus site of Ala-Gly- X, where cleavage is proposed to take place between the Gly and X and X is usually an aliphatic residue. The immunological reagents were used in conjunction with immunofluorescent and immunogold labeling analyses to identify the location of these core proteins during virion assembly. Core proteins were localized to the virosomes in VV infected cells, to the viroplasm of immature virus particles, and to the center of mature virions. Precursor specific antiserum indicated that the larger molecular weight precursors of core proteins are within immature virions as well. From these results the following conclusions can be made. Identification of a putative cleavage consensus site suggests that proteolytic processing is an endoproteolytic event. The observation that precursor structural proteins were found within immature particles indicates that the proteinase responsible for cleavage is also present. The fact that assembly has to occur before proteolytic processing of VV structural proteins suggests that the cleavage events are dependent upon a specific core protein conformation. However the nature of this conformational requirement is not known. Further research is underway to develop a full understanding of the proteolytic events during virion morphogensis

Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised