Judy K. VanSlyke

Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface.

Furin is a membrane‐associated endoprotease that efficiently cleaves precursor proteins on the C‐terminal side of the consensus sequence, Arg‐X‐Lys/Arg‐Arg1, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C‐terminal side of the enzymes autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi‐associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans‐Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse‐chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exist from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.

Activation of the furin endoprotease is a multiple‐step process: requirements for acidification and internal propeptide cleavage

Activation of furin requires autoproteolytic cleavage of its 83‐amino acid propeptide at the consensus furin site, Arg‐Thr‐Lys‐Arg107↓. This RER‐localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER‐localized soluble furin construct to either: (i) an acidic and calcium‐containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co‐immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (‐Arg70‐Gly‐Val‐Thr‐Lys‐Arg75↓‐). The significance of this cleavage in regulating the compartment‐specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.

Regulation of Connexin Degradation as a Mechanism to Increase Gap Junction Assembly and Function

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t 1 2 = 1.5–5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.

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.

Conformational Maturation and Post-ER Multisubunit Assembly of Gap Junction Proteins

For all previously well-characterized oligomeric integral membrane proteins, folding, multisubunit assembly, and recognition of conformationally immature molecules for degradation occurs at their organelle of synthesis. This cannot, however, be the case for the gap junction-forming protein connexin43 (Cx43), which when endogenously expressed undergoes multisubunit assembly into connexons only after its transport to the trans-Golgi network. We have developed two novel assays to assess Cx43 folding and assembly: acquisition of resistance of disulfide bonds to reduction by extracellularly added DTT and Triton X-114 detergent phase partitioning. We show that Cx43 synthesized at physiologically relevant levels undergoes a multistep conformational maturation process in which folding of connexin monomers within the ER is a prerequisite for multisubunit assembly in the TGN. Similar results were obtained with Cx32, disproving the widely reported contention that the site of endogenous beta connexin assembly is the ER. Exogenous overexpression of Cx43, Cx32, or Cx26 allows these events to take place within the ER, the first example of the TGN and ER as alternative sites for oligomeric assembly. Our findings also constitute the first biochemical evidence that defective connexin folding is a cause of the human disorder X-linked Charcot-Marie-Tooth disease.

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Using cultured lens epithelial cells, we discovered a new type of cross-talk between the FGF and TGF-β pathways, as well as a novel role for TGF-β and p38 kinase in the regulation of gap junctional intercellular communication. Our findings provide an explanation for how pathologically increased TGF-β signaling could contribute to cataract formation.

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis.

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.

[4] Use of vaccinia virus vectors to study neuropeptide processing

Publisher Summary This chapter discusses the use of vaccinia virus vectors to study neuropeptide processing. The construction of a vaccinia recombinant is rapid and not difficult. For example, purified recombinant virus can be prepared in less than two weeks. The vaccinia genome can accommodate large and/or multiple DNA inserts. Foreign genes are inserted by homologous recombination, precluding clonal variations. High levels of foreign protein can be obtained routinely by cloning the cDNAs along with vaccinia promoters. Infection with vaccinia is efficient. Between 80 and 100% of the cells in a population can be made to express the foreign protein. The broad host range of vaccinia allows information to be shuttled readily and rapidly between a variety of mammalian cell types and species. Importantly, experiments can be performed with primary cultures as well as established cell lines. Because cells can be infected simultaneously with multiple vaccinia recombinants, interactions among a group of foreign proteins can be studied in a defined cell system. Finally, vaccinia expression experiments can be performed within 24 hr.

[21] Preparation of recombinant vaccinia virus for expression of small GTPases

Publisher Summary This chapter describes a method for making a vaccinia virus (VV) expression vector using the neomycin resistance gene in the recombinant plasmid pZVNEO and the selection of the recombinant VV with the antibiotic G418 sulfate (Geneticin, GIBCO-BRL). The foreign gene is being inserted behind the VV 7.5K promoter that has been used in most heterologous VV expression vectors because it contains early and late promoter elements and directs the efficient expression of the gene of interest throughout the viral life cycle. As an example, the chapter presents the generation of a recombinant VV expressing the vesicular stomatitis virus glycoprotein (VSV-G) protein that has been extensively used as a marker protein to study protein transport through the secretory pathway (Dascher and Balch, and references therein). The chapter also describes procedures for growing, isolating, and manipulating VV to familiarize the reader with basic techniques that are used in the process of isolating the recombinant virus.