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.

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Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

The Journal of Cell Biology ◽

10.1083/jcb.115.5.1357 ◽

1991 ◽

Vol 115 (5) ◽

pp. 1357-1374 ◽

Cited By ~ 495

Author(s):

L S Musil ◽

D A Goodenough

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Gap Junction ◽

Intracellular Transport ◽

Surface Protein ◽

Immunohistochemical Localization ◽

Junctional Communication ◽

Junction Protein ◽

Gap Junctional ◽

Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

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Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.10.6.2033 ◽

1999 ◽

Vol 10 (6) ◽

pp. 2033-2050 ◽

Cited By ~ 143

Author(s):

Karen Jordan ◽

Joell L. Solan ◽

Michel Dominguez ◽

Michael Sia ◽

Art Hand ◽

...

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Gap Junctions ◽

Gap Junction ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Mdck Cells ◽

Rat Kidney ◽

Dye Transfer ◽

Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

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Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins.

The Journal of Cell Biology ◽

10.1083/jcb.105.3.1227 ◽

1987 ◽

Vol 105 (3) ◽

pp. 1227-1240 ◽

Cited By ~ 120

Author(s):

S A Green ◽

K P Zimmer ◽

G Griffiths ◽

I Mellman

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Golgi Apparatus ◽

Cell Surface ◽

Intracellular Transport ◽

Rat Kidney ◽

Fc Receptor ◽

Lysosomal Membrane ◽

Plasma Membrane Proteins ◽

Kinetics Of

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

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The origin of annular junctions: a mechanism of gap junction internalization

Journal of Cell Science ◽

10.1242/jcs.114.4.763 ◽

2001 ◽

Vol 114 (4) ◽

pp. 763-773 ◽

Cited By ~ 15

Author(s):

K. Jordan ◽

R. Chodock ◽

A.R. Hand ◽

D.W. Laird

Keyword(s):

Cell Surface ◽

Gap Junction ◽

Fluorescent Protein ◽

Rat Kidney ◽

Time Lapse ◽

Gap Junctional Intercellular Communication ◽

Gap Junction Channels ◽

Normal Rat ◽

Connexin Proteins ◽

Green Fluorescent

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3–6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

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Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.2.c302 ◽

1993 ◽

Vol 264 (2) ◽

pp. C302-C310 ◽

Cited By ~ 89

Author(s):

H. Birn ◽

J. Selhub ◽

E. I. Christensen

Keyword(s):

Plasma Membrane ◽

Proximal Tubule ◽

Brush Border ◽

Intracellular Transport ◽

Brush Border Membrane ◽

Specific Binding ◽

Binding Protein ◽

Rat Kidney ◽

Folate Binding Protein

Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.

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Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells.

The Journal of Cell Biology ◽

10.1083/jcb.94.1.28 ◽

1982 ◽

Vol 94 (1) ◽

pp. 28-35 ◽

Cited By ~ 93

Author(s):

E G Hayman ◽

A Oldberg ◽

G R Martin ◽

E Ruoslahti

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Chondroitin Sulfate ◽

Rat Kidney ◽

Heparan Sulfate Proteoglycan ◽

Chondroitin Sulfate Proteoglycan ◽

Transformed Cells ◽

Kidney Cells ◽

Sulfate Proteoglycan ◽

Normal Rat

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

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TGN38/41 recycles between the cell surface and the TGN: brefeldin A affects its rate of return to the TGN.

Molecular Biology of the Cell ◽

10.1091/mbc.4.1.93 ◽

1993 ◽

Vol 4 (1) ◽

pp. 93-105 ◽

Cited By ~ 75

Author(s):

B Reaves ◽

M Horn ◽

G Banting

Keyword(s):

Plasma Membrane ◽

Specific Binding ◽

Rat Kidney ◽

Brefeldin A ◽

Integral Membrane Protein ◽

Intracellular Compartments ◽

Normal Rat ◽

Golgi Network ◽

Lumenal Domain ◽

Kinetics Of

TGN38 and TGN41 are isoforms of an integral membrane protein (TGN38/41) that is predominantly localized to the trans-Golgi network (TGN) of normal rat kidney cells. Polyclonal antisera to TGN38/41 have been used to monitor its appearance at, and removal from, the surface of control and Brefeldin A (BFA)-treated cells. Antibodies that recognize the lumenal domain of TGN38/41 are capable of specific binding to the surface of both control and BFA-treated cells. In both control and BFA-treated cells internalized TGN38/41 is targeted to the TGN; however, there are differences in 1) the morphology of the intracellular structures through which TGN38/41 passes and 2) the kinetics of internalization. These data demonstrate that TGN38/41 cycles between the plasma membrane and the TGN in control and BFA-treated cells and suggest that recycling pathways between the plasma membrane and the TGN exist for predominantly TGN proteins as well as those that normally cycle to other intracellular compartments. They also demonstrate that addition of BFA not only alters the morphology and localization of the TGN but also the kinetics of endocytosis.

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Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

10.1101/211607 ◽

2017 ◽

Cited By ~ 1

Author(s):

Rachael M. Kells-Andrews ◽

Rachel A. Margraf ◽

Charles G. Fisher ◽

Matthias M. Falk

Keyword(s):

Plasma Membrane ◽

Gap Junctions ◽

Gap Junction ◽

Connexin 43 ◽

Room Temperature ◽

Cell Communication ◽

Mapk Phosphorylation ◽

Annular Gap ◽

Summary Statement ◽

Triton X

ABSTRACTGap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.Summary StatementHere we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.Abbreviations usedAGJannular gap junctionAMSHassociated molecule with the SH3 domain of STAMCMEclathrin-mediated endocytosisCxConnexinCx43Connexin 43DUBdeubiquitinaseGJgap junctionMonoUbmonoubiquitinNedd4-1neural precursor cell expressed developmentally down-regulated protein 4-1PMplasma membranePolyUbpolyubiquitinTPA12-O-Tetradecanoylphorbol 13-AcetateTX-100Triton X-100RTroom temperatureUbubiquitin

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TRAIL Induces Nuclear Translocation and Chromatin Localization of TRAIL Death Receptors

Cancers ◽

10.3390/cancers11081167 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1167 ◽

Cited By ~ 1

Author(s):

Ufuk Mert ◽

Alshaimaa Adawy ◽

Elisabeth Scharff ◽

Pierre Teichmann ◽

Anna Willms ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Nuclear Export ◽

Nuclear Translocation ◽

Nuclear Accumulation ◽

Nuclear Trafficking ◽

Surface Receptors ◽

Tnf Superfamily ◽

Cell Surface Biotinylation ◽

Intracellular Compartments

Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.

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Gap junction Heterogeneity in Liver, Heart, and Lens

Physiology ◽

10.1152/physiologyonline.1988.3.5.206 ◽

1988 ◽

Vol 3 (5) ◽

pp. 206-211

Author(s):

PG FitzGerald

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Molecular Analysis ◽

Tissue Specificity ◽

Structure And Function ◽

Junctional Proteins ◽

And Function ◽

Junction Structure

Gap junctions are nearly ubiquitous structures that ionically and metabolically couple adjacent cells. Molecular analysis of junctional proteins is establishing the presence of families of unique but homologous junctional proteins, opening the door to an explanation of tissue specificity in gap junction structure and function.

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