Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER

Cell, Vol. 74, 1065-1077,

September

24, 1993, Copyright

0 1993 by Cell Press

Multisubunit Assembly of an Integral Plasma Membrane Channel Protein, Gap Junction Connexin43, Occurs after Exit from the ER Linda S. Musk and Daniel A. Goodenough Department of Cell Biology Harvard Medical School Boston, Massachusetts 02115

Summary Connexin43 (Cx43) is an integral plasma membrane protein that forms gap junctions between vertebrate cells. We have used sucrose gradient fractionation and chemical cross-linking to study the first step in gap junction assembly, oiigomerization of Cx43 monomers into connexon channels. in contrast with other plasma membrane proteins, multisubunit assembly of Cx43 was specifically and completely blocked when endoplasmic reticulum (ER)-to-Golgi transport was inhibited by 15% incubation, carbonyi cyanide mthlorophenyihydrazone, or brefeidin A or in CHOceil mutants with temperature-sensitive defects in secretion. Additional experiments indicated that connexon assembly occurred intraceiiuiarly, most likely in the trans-Goigi network. These results describe a post-ER assembly pathway for integral membrane proteins and have implications for the relationship between membrane protein oligomerization and intracellular transport. Introduction Oligomerization is an important and common posttranslational modification of integral plasma membrane proteins (Hurtley and Helenius, 1989). In addition to its role in protein function, oligomerization is thought to be a critical determinant of transport of nascent membrane proteins along the secretory pathway. From studies of both viral and cellular membrane proteins, the concept has emerged that subunits of integral plasma membrane proteins undergo specific multisubunit assembly in the endoplasmic reticulum (ER) before they can be transported through the Golgi complex and ultimately to the cell surface (Rose and Doms, 1988; Hurtley and Helenius, 1989; Gething et al., 1988). Free subunits (or those incorporated into incompletely or improperly assembled complexes) are generally degraded either within the ER or in lysosomes and do not reach the plasma membrane. This process is part of the so-called quality control (Hurtley and Helenius, 1989) that prevents expression of abnormal or otherwise inappropriate proteins on the cell surface. We have studied the multisubunit assembly of gap junctions, plasma membrane specializations that mediate the regulatable transfer of small molecules and ions between adjoining cells. As the principal means by which direct cell-cell communication is achieved in animal tissues, gap junctions are thought to be essential for signal propagation in electrically excitable cells (De Mello, 1987) and to be involved in growth control, differentiation, and embryonic development (Loewenstein and Rose, 1992; Guthrie and

Gilula, 1989). Research over the last several years has revealed that gap junctions are comprised of members of a multigene family of nongiycosylated integral plasma membrane proteins, the connexins (reviewed in Bennett et al., 1991). As inferred from structural studies of isolated gap junctions (see Caspar et al., 1988), assembly of gap junctions is a multistage process. The first step in junction formation is thought to be oiigomerization of six (or five; L. S. M., D. A. G., and L. Makowski, unpublished data) connexin molecules into a membrane channel called a connexon. Next, a connexon in the plasma membrane of one cell must pair with a connexon in an apposing cell membrane to form an intercellular channel. These channels become densely clustered at cell-cell interfaces into gap junctional plaques in which connexins are the only detectable proteins. We have used a combination of biochemical and genetic techniques to analyze the assembly of connexin43 (Cx43), thought to be the first gap junction protein expressed by the zygotic genome during mouse embryogenesis (Valdimarsson et al., 1991) and a mediator of cell-cell communication in a wide variety of adult organs (e.g., heart, uterus, brain, lens, and kidney) (Beyer et al., 1989). Previous studies have demonstrated that assembly of Cx43 into gap junctional plaques is tightly correlated with Cx43 phosphorylation and acquisition of insolubility in Triton X-100, both of which are dependent on intercellular adhesion mediated by cell-cell adhesion molecules(Musil et al., 1990b; Musil and Goodenough, 1991). In the current study, we have analyzed the process of assembly of Cx43 monomers into connexons. As assessed by both sucrose gradient velocity sedimentation and chemical cross-linking, connexon assembly occurred intracellularly but was completely blocked when ER-to-Golgi transport was disrupted by any of four independent methods. These findings provide compelling evidence that oligomerization of Cx43 into connexons occurs after exit from the ER and distinguish connexin assembly from that of other well-characterized integral plasma membrane proteins for which assembly in the ER is a prerequisite for transport through the secretory pathway (reviewed in Rose and Doms, 1988; Hurtley and Helenius, 1989). These results have important implications for gap junction biosynthesis and for the mechanism of quality control of newly synthesized integral plasma membrane proteins. Results Sucrose Gradient Velocity Sedimentation Analysis of Connexon Assembly We have previously shown that in NRK fibroblasts and other cells, newly synthesized Cx43 is soluble in 1% Triton X-100 detergent at 4OC and acquires resistance to Triton solubilization only upon incorporation into gap junctional plaques (Musil and Goodenough, 1991). This finding raised the possibility that connexons are Triton soluble and that their assembly from free connexin monomers

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1. Sucrose

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(A and B) NRK cell monolayers were metabolically labeled for 5 hr with [%]methionine at either 37OC or 20% and then lysed at 4OC in the presence of 1% Triton X-100. Triton-insoluble gap junctional plaques were removed from the cell lysates by centrifugation at 100,000 x g, and the supernatants were fractionated on 5%-20% linear sucrose gradients as described in Experimental Procedures. Equal fractions were collected, assayed for sucrose content, and then immunoprecipitated with affinity-purified antiCx43 antibodies. Each immunoprecipitate was analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography (A). The amount of Cx43 in each gradient fraction was quantitated by laser densitometry and expressed relative to the Cx43 signal in the fraction with the highest P%]methionine-Cx43 content (B). The upper band of the Cx43 doublet seen at the top in (A) represents the form of phosphorylated Cx43 that is partially soluble in Triton X-100 at 4OC (Cx43-P,). No detectable phosphorylation of Cx43 occurs at 20°C ([A], bottom) (Musil and Goodenough, 1991). The slight difference in the 5s region between the two gradient profiles seen in (B) is not reproducible or significant. (C and D) Sucrose gradient fractionation profiles of [Yjjmethionine-Cx43 translated in vitro (IVT) in the absence of membranes (Musil et al., 199Oa) ([Cl, closed triangles); [“Slmethionine-Cx43 from NRK cell lysates boiled in 0.6% SDS prior to sucrose gradient fractionation ([Cl, open diamonds); and [“Sjmethionine-Cx43 from NRK cells pulsed for 20 min (20’ P) at 37°C and lysed in Triton X-100 either immediately after labeling ([Cl, closed circles) or after a 2 hr chase at 37OC (D).

could be examined by velocity centrifugation (Figure 1). The experimental strategy used was modeled after that developed for study of the assembly of another oligomeric integral membrane channel protein, the nicotinic acetylcholine receptor (nAchR) (Blount and Merlie, 1988). Since sedimentation rate in a 5%-200/o sucrose gradient is largely based on molecular mass and shape (Rickwood and Chambers, 1984) it seemed likely that the sedimentation coefficient of monomeric Cx43 (M,, 43 kd) in such a gradient would be similar to that of unassembled nAchR a subunit (M,, 42 kd; 5S), whereas connexons would be expected to migrate near the position of the nAchR pentamer (M,, 250 kd; 9s). As shown at the top of Figure 1A, Triton-soluble lysates prepared from NRK cells metabolically labeled with [YSlmethionine at 37OC for 5 hr could be fractionated into two distinct peaks of immunoprecipitable Cx43 in 5%-20% linear sucrose gradients. The major peak of [35S]methionine-Cx43 recovered was centered at 9%-100/a sucrose and comigrated with a 5S standard, at approximately the same position as that reported for unassembled nAchR a subunit subjected to centrifugation under identical conditions. A second peak of Cx43 immu-

noreactivity migrated consistently at 15%-l 6% sucrose, in the position of assembled nAchR (9s) (see Blount and Merlie, 1988). In contrast, [35S]methionine-Cx43 from NRK cells labeled for only 20 min was recovered exclusively in the 5S peak (Figure lC, closed circles) and comigrated with monomeric Cx43 generated either by SDS denaturation of NRK cell lysates (Figure lC, open diamonds) or by in vitro translation of rat Cx43-encoding messenger RNA (mRNA) in a membrane-free reticulocyte lysate (Figure lC, closed triangles). If, however, cells were pulsed for 20 min and then chased for 2 hr at 37OC prior to sucrose gradient fractionation, a peak of (VlmethionineCx43 was also detected at 9s (Figure lD), as expected if there were a precursor-product relationship between the 5S monomer and 9S oligomer populations. Both the 5s and 9S [Y9]methionine-Cx43 peaks were also obtained when NRK cells were metabolically labeled for 5 hr at 20°C (Figure lA, bottom; Figure lB, open circles), conditions under which newly synthesized Cx43 remains Triton soluble and is not incorporated into gap junctional plaques (Musil and Goodenough, 1991). Recentrifugation of the 5S and 9S peaks on separate 5%-20% sucrose gradients

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Triton-soluble lysates were prepared from NRK cells labeled with [%Sjmethionine at 37OC for 5 hr and fractionated on a 5%-20% linear sucrose gradient as in Figure 1. The fractions containing the 5S (8%11% sucrose; lanes 1 and 2) and the 9S (14%-17% sucrose; lanes 3-5) Cx43 species were collected and incubated separately with either 506 ug/ml DSP (lanes 2,4, and 5) or an equal volume of DMSO only (lanes 1 and 3). Cx43 was immunoprecipitated and analyzed on a 3%14% polyacrylamide gradient gel in either the absence (lanes l-4) or presence (lane 5) of f3-mercaptoethanol. Lane C is the same as lane 4, except immunoprecipitated with an irrelevant antibody. The band migrating at - 400 kd in lane 4 is probably a specifically immunoprecipitated connexon dimer.

did not change their sedimentation properties, suggesting that the two Cx43 populations were stable and did not interconvert during sucrose gradient fractionation (data not shown). Cross-Linking Analysis of Connexon Assembly The composition of the 9s Cx49containing complex was examined by chemical cross-linking with the thiol-cleavable homobifunctional reagent dithiobis(succinimidyl propionate) (DSP) (Figure 2). [35S]methionine-labeled NRK cell lysates were fractionated on linear sucrose gradients as described above, and the pooled fractions containing the 9s Cx43 population were incubated in either the presence or absence of 500 uglml DSP for 30 min at 4OC. DSP efficiently cross-linked [35S]methionine-Cx43 in the 9s peak from an -40 kd monomer (Figure 2, lane 3) to a species that migrated at -200 kd (lane 4). Although reproducible, this 200 kd value cannot be taken as an accurate measure of the molecular mass of the complex since Cx43, like many other proteins (Staros and Anjaneyulu, 1969) migrates anomalously fast on SDS-polyacryl-

amide gel8 after cross-linking, especially in the absence of reducing agent (compare lanes 1 and 2 in Figure 2). Reduction of DSP-induced cross-links with 6-mercaptoethanol prior to SDS-polyacrylamide gel electrophoresis quantitatively converted the 200 kd species to monomeric Cx43, without detectable recovery of other labeled proteins (Figure 2, lane 5). Neither the 200 kd oligomer nor larger aggregate8 were formed when sucrose gradient fractions containing the 5s (monomer) population of Cx43 (8%-l 1% sucrose) were cross-linked identically (Figure 2, lane 2). Cross-linking of Cx43 to the 200 kd species did not require prior sucrose gradient fractionation of the cell lysate. In Figure 3A, [35S]methioninelabeled NRK cells were lysed in the presence of Triton X-100 at 4OC and the solubilized material incubated directly with DSP. Both the monomeric (40 kd) and 200 kd forms of Cx43 were immunoprecipitated (Figure 3A, lane 3). The extent of Cx43 cross-linking was dependent on the concentration of DSP used (maximum recovery of the 200 kd complex with 50-100 uglml DSP) and was unchanged over a 7-fold range of lysate concentration (compare lanes 3 and 5 in Figure 3A), indicating that cross-linking to the 200 kd form was dilution resistant. Similar results were obtained when another homobifunctional cross-linker, ethylene glycolbis(succinimidyl succinate) (EGS), was substituted for DSP (Figure 3A, lane 8). Cross-linked 200 kd Cx43 migrated on a linear sucrose gradient in asingle peak at 15%-16% sucrose, confirming its identity as the 9s form of Cx43 (Figure 38). Cx43 in the gap junction-associated (i.e., Triton-resistant at 4OC) fraction could also be partially cross-linked to the 200 kd form, provided the junctions had first been solubilized by warming to 25OC in the presence of Triton X-100 (Musil and Goodenough, 1991) (Figure 3C). On the basis of these results and the findings presented in Figures 1 and 2, we conclude that the 9s 200 kd species is a stable, specific noncovalent homo-oligomer of Cx43 that is assembled into gap junctional plaques and is therefore an authentic connexon. Connexon Assembly in NRK Cells Is Blocked By inhibitors of ER-to-Golgi Transport To determine where connexon formation takes place, movement of newly synthesized Cx43 through the secretory pathway was arrested at distinct sites with known inhibitors of intracellular transport. The assembly state of Triton-soluble Cx43 was then assessed using sucrose gradient rate sedimentation and chemical cross-linking as independent assays of connexon formation (Figure8 4-7). Incubation of mammalian cells at temperatures under 16OC prevents the transport of newly synthesized secretory and membrane proteins to the Golgi, presumably by causing their accumulation in a tubulovesicular membrane system between the ER and the Golgi that has been referred to a8 the intermediate compartment (Saraste and Kuismanen, 1984; Schweizer et al., 1990). NRK cells were metabolically labeled at 15’C for 5 hr, and the [YS]methionine-Cx43 synthesized during this period was analyzed by both sucrose gradient fractionation (Figure 4A, closed squares) and DSP cross-linking (Figure 48, lane 2). No

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(A) NRK cells labeled for 5 hr at 20°C were lysed in the presence of 1% Triton X-100, and the solubilized extracts were incubated with DMSO only (lanes 1 and 2) 100 ug/ml DSP (lanes 3-7) or 500 uglml EGS (lane 8) without prior sucrose gradient fractionation. The sample in lane 5 was diluted 7-fold with Triton-containing buffer before cross-linking. Samples were immunoprecipitated with either anti-Cx43 antibodies (lanes 1, 3, 5, 8, and 8) or with an irrelevant affinity-purified antibody preparation (lanes 2, 4, and 7) and analyzed on 4%-15% polyacrylamide gradient gels under nonreducing conditions (lanes l-5 and 8) or after reduction with 8-mercaptoethanol (BME) (lanes 8 and 7). Qualitatively similar results were obtained if the cells were labeled at 37OC instead of 20°C. Sands migrating at >380 kd (also seen in Figures 4-6) are nonspecifically immunoprecipitated (see lane 2) and probably represent binding to the protein A-Sepharose immunoadsorbant of fibronectin (Doran and Raynor, 1981) large quantities of which are synthesized by certain NRK cells (Louvard et al., 1982). (8) NRK cell extracts prepared as in (A) were cross-linked with 100 @ml DSP, incubated with 30 mM glycine to quench the cross-linking reaction, and then fractionated on a 5%-200/p linear sucrose gradient exactly as described in Figure 1. Cx43 was immunoprecipitated from each fraction and analyzed on a 4%-15% gel in the absence of reducing agent. (C) NRK cells labeled with PS]methionine for 5 hr at 37OC were lysed at 4OC with 1% Triton X-100, after which the Triton-insoluble gap junctions were collected by centrifugation at 100,000 x g for 50 min. The pellet was resuspended in incubation buffer containing 1% Triton X-100, incubated at 25OC for 30 min to solubilize gap junctions partially, and then subjected to a second 100.000 x g centrifugation. This supernatant was analyzed by cross-linking with 100 ug/ml DSP followed by immunoprecipitation with antiCx43 antibodies. Molecular masses of protein standards are given in kilodaltons.

multimeric forms of Cx43 were detected by either assay. If ceils labeled at 15% were chased for 2 hr at 20% prior was reto cell lysis, - 40% of the [35S]methionine-Cx43 covered as connexons (Figure 4A, open circles; Figure 48, lane 4) whereas conversion to connexons was negligible if the cells were chased at 15% for the same period (data not shown). Thus, Cx43 synthesized during the 15% block was not irreversibly converted to an assembly-incompetent form during retention in the intermediate compartment or the ER. To rule out the possibility that assembly of newly synthesized Cx43 was thermodynamically unfavorable at 15% rat Cx43 was expressed in Xenopus oocytes maintained at this temperature (Figures 4C and 4D). Unlike mammalian cells, Xenopus oocytes efficiently transport newly synthesized proteins to the plasma membrane at <20°C (Colman, 1984) and form functional gap junctional channels upon pairing even at 14% (Ft. L. Gimlich and D. A. G., unpublished data). Synthetic RNAencoding rat Cx43 (the same protein that is endogenously expressed in NRK cells) was microinjected into stage VI Xenopus oocytes kept at 15%. Connexon formation was consistently detected by both sucrose gradient fractionation (Figure 4C) and by chemical cross-linking (Figure 40, lane 2) demonstrating that rat Cx43 can oligomerize

at 15% when expressed in a system in which ER-to-Golgi transport continues, but cannot in NRK cells in which such movement is inhibited. Cell surface biotinylation experiments conducted in parallel with the temperature shift studies shown in Figures 4A and 4B demonstrated that none of the [%]methionineCx43 synthesized during a 5 hr pulse at 15OC was present on the cell surface after a 2 hr chase at 20% (data not shown; see Musil and Goodenough, 1991). This is consistent with previous studies demonstrating that incubation of mammalian cells at 20% blocks the intracellular transport of nascent secretory and integral membrane proteins within the trams-Golgi network (TGN) for at least 2 hr (Saraste and Kuismanen, 1984; Griffiths and Simons, 1988). Despite the lack of cell surface Cx43, NRK cells subjected to this pulse-chase protocol did, however, assemble large amounts of [%]methionine-Cx43 into connexons (Figure 4A, open circles; Figure 48, lane 4). We conclude that connexon oligomerization occurs intracellularly, although whether all Cx43 molecules are assembled prior to transport to the plasma membrane is not known. The absence of connexon formation in NRK cells at 15% distinguished the assembly of Cx43 from that of several other integral plasma membrane proteins (see Table

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Figure 4. Connexon Assembly Is Blocked at 15% in NRK Cells but Not in Xenopus Oocytes (A and 8) NRK cells were metabolically labeled with [%]methionine for 5 hr at WC without a chase ([A], closed squares; [B], lanes 1 and 2) or with a 2 hr chase at 20% ([A], open circles; [B], lanes 3 and 4). Triton-soluble lysates were prepared from the cells and analyzed for connexon assembly using either sucrose gradient fractionation (A) or cross-linking(B) followed by immunoprecipitation with antiCx43 antibodies. In (B), lanes 2 and 4 were incubated with 100 @ml DSP, and lanes 1 and 3 are uncrosslinked (DMSO only) controls. (C and D) Stage VI Xenopus oocytes were injected with rat Cx43encoding cRNA and then labeled for 5 hr at 15% with PS]methionine. Triton-soluble lysates were prepared and then assayed by sucrose gradient fractionation (C) or DSP cross-linking (D) exactly as described for NRK cells. In (D), lanes 2 and 3 were crosslinked with DSP (100 uglml), and lane 1 was mock cross-linked with DMSO only. Lanes 1 and 2 were immunoprecipitated with antiCx43 antibodies and lane 3 with an irrelevant antibody preparation. A sample of DSP crosslinked Cx43 from NRK cells is included (NRK) and comigrates with Cx43 from Xenopus oocytes (lane 2).

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1) and was consistent with a post-ER site of connexon oligomerization. Cx43 assembly was also inhibited by the ER-to-Golgi transport blocker carbonyl cyanide m-chlorophenylhydrazone (CCCP) (see Figure 7, lane 7; data not shown for NRK cells). Interpretation of the latter result is complicated, however, by the fact that CCCP severely reduces intracellular ATP levels and protein synthesis and will thus inhibit any process dependent on either metabolic energy or ongoing protein production regardless of intracellular location (Tartakoff and Vassalli, 1979; see also Discussion). We therefore examined the effect on Cx43 assembly of brefeldin A (BFA), a fungal antibiotic that inhibits ER-to-Golgi trafficking without appreciably reducing either protein synthesis or ATP levels (Misumi et al., 1988) (Figure 5). BFA inhibits the transport of newly synthesized proteins to the Golgi and induces the vesiculation of cis, medial, and trans-Golgi cisternae and their fusion with the ER in a microtubule-dependent process (LippincottSchwartz et al., 1990; Klausner et al., 1992). Although BFA did not significantly inhibit Cx43 synthesis in NRK cells, nascent [%]methionine-Cx43 remained unphosphorylated (Figure 58, inset) and did not acquire resistance to Triton solubilization (data not shown), indicating that transport of Cx43 to the cell surface was blocked. In addition, the perinuclear Golgi-like staining pattern normally observed for Cx43 by immunofluorescence (Figure 5A; see also Musil et al., 1990b; Musil and Goodenough, 1991) was lost in BFA-treated cells and replaced by a diffuse reticular antiCx43 reactivity (Figure 58) typical of proteins redistributed to the fused ER-Golgi compartment

1 2 3

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in NRKcells(Lippincott-Schwartzet al., 1989,1990,1991; Wood et al., 1991; Reaves and Banting, 1992). Both the inhibition of Cx43 processing and the loss of Golgiassociated Cx43 staining were fully reversible when BFA was removed (Figure 5C). Taken together, these results indicated that BFA affected the intracellular transport of Cx43 in a manner similar to that described for other newly synthesized secretory and integral membrane proteins. Analysis of lysates of BFA-treated NRK cells either by sucrose gradient fractionation (Figure 5D, closed squares) or by DSP cross-linking (Figure 5E, lane 4) yielded no detectable connexon-assembled (35S]methionine-Cx43, even if the DSP concentration was increased 1 O-fold (data not shown). BFA also reversibly blocked connexon formation in the presence of 20 @ml nocodazole (Figure 5D, closed circles; Figure 5E, lane 7), which has been reported to inhibit (although not to block completely) BFA-induced fusion of Golgi-derived vesicles with the ER in NRK cells by depolymerizing microtubules (Lippincott-Schwartz et al., 1990). In contrast, nocodazole alone had little effect on connexon assembly (data not shown). Lack of connexon assembly in the presence of BFA is therefore likely to be due to the absence of normal ER-to-Golgi traffic rather than to the fusion and subsequent mixing of ER and Golgi components. Connexon Assembly in CHO Cell Intracellular Transport Mutants Krieger and colleagues (A. Fisher, L. Hobbie, S. Lee, and M. Krieger, submitted; see also Fisher, 1992) havecharac-

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(A-C) NRK cultures were incubated at 37% for 5 hr in the absence (A) or presence (B and C) of 6 uglml BFA (GIBCO BRL, Grand Island, New York). In (C), cultures were chased for an additional 1.5 hr in BFA-free medium. All cells were fixed and processed for immunofluorescence using antiCx43 antibodies, as described in Experimental Procedures. The insets show total cellular Cx43 immunoprecipitated from duplicate cultures metabolically labeled with [%]methionine at 37V for 5 hr either without BFA (A), with BFA (B), or with BFA followed by a 1.5 hr chase without BFA (C). The slower-migrating Cx43 bands (A and C) are due to phosphorylation of Cx43 after transport to the cell surface (Musil and Goodenough, 1991) and are not produced in the presence of BFA (B). (D and E) NRK cells were incubated in methionine-free labeling medium with ([D], closed squares and closed circles; [Ej, lanes 3-7) or without (ID], open squares; [El, lanes 1 and 2) 6 @ml BFA for 30 min prior to the addition of j%]methionine to the tissue culture dishes. After 5 hr at 37% Triton-soluble lysates were prepared and subjected to sucrose gradient fractionation (D) or cross-linking (E). In some cases (ID], closed circles; [El, lanes 6 and 7). cells were incubated with 20 hg/ml nocodazole (NOC) for 40 min prior to addition of BFA and labeled in the continuous presence of both drugs. In (E), minus denotes samples mock cross-linked in the absence of DSP, and lanes marked plus show parallel samples reacted with 100 ug/ml DSP. All samples were immunoprecipitated with antiCx43 antibodies with the exception of lane 5. which is a preimmune control.

terized CHO cell mutants with temperature-sensitive defects in the secretory pathwy. These cells express Cx43 and therefore provide a novel system in which the intracellular transport of Cx43 can be manipulated nonpharmacologically. We have examined connexon assembly in the two classes of CHO mutants (Id/F and Id/G) showing the most severe defects in protein secretion at the nonpermissive temperature (39’%-40.5%). As reported by Fisher et al. (submitted), functions localized to the ER appear to be normal in both /d/F and /d/G cells even at the restrictive temperature, including assembly of a multisubunit integral plasma membrane protein (the scavenger receptor). However, in MFcells maintained at 40% for 7-12 hr, oligosaccharides of all four of the integral plasma membrane glycoproteins they studied fail to be detectably processed to mature forms under the labeling conditions tested. Only

one of these glycoproteins (the low density lipoprotein receptor) acquires, abnormally slowly, detectable resistance to endoglycosidase H, a process localized to the medial Golgi. The most proximal transport defect in /d/F cells therefore appears to be before the trans cisternae of the Golgi complex and is most likely somewhere between the ER and the medial Golgi. The phenotype of mutants in the /d/G complementation group is not as severe as that of the /d/F mutants, and nascent glycoproteins are transported, albeit very slowly, to the trans Golgi as evidenced by low but detectable processing of the low density lipoprotein receptor to the mature form (Fisher, 1992). In the experiment shown in Figure 6, /d/F, Id/G, and wildtype CHO cells were either maintained at the permissive temperature (34%) or shifted to the restrictive temperature (40%) for 12 hr to allow full expression of the mutant

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Newly confluent wild-type (lanes l-5) /d/F (lanes 6-9) or /d/G (lanes 1 O-l 3) CHO cells were incubated for 12 hr at either the permissive temperature (34OC; lanes l-3,6,7, 10, and 11) or the restrictive temperature @WC; lanes 4,5,6,9, 12, and 13) prior to metabolic labeling with [“SJmethionine for 4 hr at the same temperature. All cells were lysed with 1% Triton X-100, and Triton-soluble extracts were subjected to cross-linking with 100 uglml DSP (lanes labeled plus) or mock cross-linked with DMSO (lanes marked minus). All samples were immunoprecipitated with antiCx43 antibodies except lane I, which is a preimmune serum control. Lanes IO-13 are overexposed relative to lanes 6-9 to facilitate visualization of the 200 kd connexon band in lane 13. No connexon assembly was detectable in /d/F cells at 40% at any exposure. The metabolic half-life of Cx43 was similar in all three cell types at both 34°C and 40°C (- 2.5 hr, typical for connexins in tissue culture cells; see Musil et al., 1990b) (data not shown).

phenotypes. As assessed by chemical cross-linking with DSP, all cells assembled Cx43 into 200 kd connexons at the permissive temperature to approximately the same extent (Figure 6, lanes 3, 7, and 11). At the restrictive temperature, the recovery of cross-linked 200 kd Cx43 was not affected in wild-type CHO ceils (Figure 6, lane 5) but no connexon assembly was detected in /d/f ceils despite nearly equivalent levels of Cx43 synthesis (lane 9). The extent of connexon assembly in /d/G cells at 40% (Figure 6, lane 13) was intermediate between that observed in wild-type and in /d/f CHO cells, consistent with the incomplete ER-to-Golgi transport block in these mutants. Taken together with our observations that BFA, CCCP, and incubation at low temperature (15% and 20%) had the same effect on connexon assembly in wildtype CHO cells as they did in NRK cells (data not shown), these results strongly support the conclusion that connexon assembly in both NRK and CHO cells occurs after exit of Cx43 from the ER and prior to transport to the plasma membrane. Connexon Assembly in Gap Junction-Deficient Cell Lines In addition to communication-competent cells, Cx43 is also synthesized by certain cell lines that are either se-

verely deficient (Sl80 sarcoma cells) or completely lacking (L929 fibroblasts) in morphologically or physiologically recognizable gap junctions and that do not phosphorylate Cx43 to its mature Cx43-P2 form (see Musil et al., 1990b). Both cell lines lack functioning intercellular adhesion molecules, and expression of cell-cell adhesion moleculeencoding cDNA has been shown to be sufficient to induce Cx43 phosphorylation and the formation of gap junctions in transfected S180 cells (Mege et al., 1988; Musil et al., 1990b). To characterize connexon assembly in these communication-defective cells, confluent cultures of S180 or L929 cells were metabolically labeled with [35S]methionine for 5 hr at 37% and then subjected to DSP cross-linking (Figure 7). Both cell lines assembled [YS]methionineCx43 into complexes that were qualitatively indistinguishable from connexons recovered from NRK cells (Figure 7A, lane 2; Figure 76, lane 2), demonstrating that neither cell-cell adhesion molecule-mediated intercellular adhesion nor phosphorylation to the Cx43-P2 form is required for connexon assembly. When assayed as described for NRK cells, connexon assembly in S180 cells was inhibited by BFA, CCCP, and 15% (but not 20%) incubation (Figure 7A, lanes 4-7; 15% data not shown). In L929 fibroblasts, the low level of radiolabeling of Cx43 after a short pulse or at temperatures below 37% precluded a compa-

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Figure 7. Connexon Assembly in Cx43-Expressing, but Gap Junctional CommunicationDeficient, Cell Lines

BFA

4mldm

Confluent monolayers of SIB0 (A) or L929 (6) cells were analyzed for connexon content after labeling with [“Slmethionine for 5 hr at 37°C ([A], lanes 1-3; [B], lanes l-3), at 20% ([A], lane 4) or at 37% with 6 kg/ml BFA ([A], lane 5; [B], lane 4). Other cultures of S180 cells were labeled for 20 min at 37% followed by a 2 hr chase at the same temperature either in the presence of 15 nfvt CCCP in glucose-free medium ([A], lane 7) or without CCCP in glucosecontaining medium ([A], lane 6). Triton-soluble extracts were prepared from all cells and incubated with DSP (100 &ml) ([A], lanes 2-7; [B], lanes 2-4) or, as a negative control, DMSO only ([A], lane 1; [B], lane 1). All sampleswere immunoprecipitated with antiCx43 antibodies except for lanes 3 of (A) and (B), which were immunoprecipitated with an irrelevant antibody preparation. A lane of DSP cross-linked Cx43 from NAK cells is included (NRK) and comigrates with Cx43 complexes from S180 or L929 cells.

L929

1

rable series of experiments, but connexon assembly in these cells was clearly sensitive to BFA (Figure 78, lane 4). Thus, communication-deficient cells, like communication-competent NRK and CHO fibroblasts, assembled Cx43 after exit from the ER. Since both S180 and L929 cells transport Cx43 to the plasma membrane (Musil and Goodenough, 1991) it is very likely that Cx43 is present on the surface of both cell types in the form of individual connexons (hemichannels) whose channel would presumably have a very low probability of opening under normal conditions. The existence of unpaired connexons on the plasma membrane has been somewhat controversial but has recently been supported by electrophysiological evidence (Paul et al., 1991; DeVries and Schwartz, 1992).

Transport

Block

Intracellular of Transport

Transport

Site Block

Inhibitors

4

Post-ER Assembly of Connexons in Cells Expressing Endogenous Cx43 The unexpected finding of this study is that assembly of newly synthesized Cx43 into connexons is blocked when ER-to-Golgi transport is inhibited by any of four methods (summarized in Table 1). Each of the ER-to-Golgi transport

We have used both sucrose gradient velocity sedimentation and chemical cross-linking to analyze a key step in

of Effect of Intracellular

3

gap junction formation, assembly of connexin monomers into connexons. The close agreement between the data obtained from these two connexon assembly assays considerably strengthens the conclusions drawn from them. For example, although certain noncovalent protein complexes are known to dissociate during ultracentrifugation in sucrose gradients (Copeland et al., 1988; Doms et al., 1987; Degen and Williams, 1991) such assemblies are often detectable by cross-linking. Conversely, chemical cross-linking is dependent on the availability of suitable reactive groups on each subunit of a protein complex, a requirement not shared by rate sedimentation analysis.

Discussion

Table 1. Summary in the ER

2

on Connexon

Formation

Connexon Assembly?

Assembly

and on Assembly

of Integral

Plasma

of Integral

Membranes

Plasma

Membrane

Proteins

in ER?

20%

TGN

Yes

Yes; see 15%

BFA

Before TGN; in fused ER-Golgi compartment

No

Yes; T cell receptor (Lippincott-Schwartz et al., 1989) mannose 6-phosphate receptor (Hike et al., 1990). vesicular stomatitis virus G protein (Doms et al., 1989) F protein (Collins and Mottet, 1991)

40°C,

/d/F cells

Unknown;

15%

Intermediate

CCCP

ER

before

trans

compartment

Golgi

No

Yes; scavenger

No

Yes; vesicular stomatitis virus G protein (Doms et al., 1987) hemagglutinin (Copeland et al., 1968) mannose Bphosphate receptor (Hille et al., 1990)

receptor

(Fisher,

1992)

No

Yes; immunoglobulin M (Tartakoff and Vassalli, 1979) hemagglutinin (Copeland et al., 1988) vesicular stomatitis G protein (Domset al., 1989) F protein (Collins and Mottet,

virus 1991)

Post-ER 1073

Assembly

of an Integral

Membrane

Protein

inhibitors we have used function by independent, albeit incompletely understood, mechanisms. Thus, although all of the treatments listed in Table 1 alter multiple cellular processes, the only obvious effect they have in common is suppression of ER-to-Golgi transport. In contrast with Cx43, assembly of the majority of well-characterized plasma membrane proteins takes place exclusively in the ER (Hurtley and Helenius, 1989) and is not blocked by the inhibitors we have used (see references listed in Table 1). For certain other multisubunit proteins (e.g., von Willebrand factor, asymmetric acetylcholinesterase, corespecific lectin, the hepatitis B surface antigen), only the first stage of oligomerization (usually disulfide-linked dimer formation) occurs in the ER, with subsequent assembly taking place in one or more post-ER intracellular compartments (Wagner, 1990; Rotundo, 1984; Colley and Baenziger, 1987; Huovila et al., 1992). Cx43 clearly differs from these proteins in that not even dimer formation is detectable under any of the conditions used in which ER-toGolgi transport is blocked. Jascur et al. (1991) have suggested that assembly of dipeptidylpeptidase IV (DPPIV), an integral plasma membrane protein, is confined to the Golgi complex. This conclusion is supported primarily by the lack of detectable DPPIV dimer with incompletely processed (high mannose type) oligosaccharides, the presence of DPPIV dimers in a purified Golgi subcellular fraction, and the ability of CCCP to block dimer formation. However, given that transport of DPPIV to the Golgi is quite fast (apparent tqh,(15 min; Stieger et al., 1988) these findings are also compatible with DPPIV dimers forming in the ER as the result of an ATP-dependent (and thus CCCP-sensitive) conformational change known to precede dimerization (Matter and Hauri, 1991), but not accumulating in the ER owing to rapid export to the Golgi. CCCP has also been reported to block the conformational maturation and subsequent dimerization of the hemagglutinin-neuraminidase glycoprotein of mumps virus (Yamada et al., 1988). No such ambiguity exists in the interpretation of our data with Cx43, since both BFA and incubation at 15% reduce connexon assembly without appreciably affecting cellular ATP levels (Misumi et al., 1986; Tartakoff, 1986). The agents we have used to inhibit connexon assembly are thought to block intracellular transport at distinct steps along the secretory pathway (see Table 1). Assembly into connexons is unlikely to occur between the ER and Golgi in the so-called intermediate compartment because incubation of mammalian cells at 15% inhibits Cx43oligomerization, even though newly synthesized proteins accumulate in this structure at <16% (Saraste and Kuismanen, 1984; Schweizer et al., 1990). In the presence of BFA, the cis, medial, and trans cisternae of the Golgi complex fuse with the ER, but the TGN does not (Chege and Pfeffer, 1990; Lippincott-Schwartz et al., 1991; Reaves and Banting, 1992; Wood et al., 1991). The inhibition of connexon assembly by BFA despite the fact that many Golgiassociated processes continue within the mixed ER-Golgi compartment is consistent with oligomerization of Cx43 occurring in the TGN. Sensitivity to BFA has been used as evidence supporting the TGN as the site of other post-

translational processing events (Shite et al., 1990; Young et al., 1990; Spiro et al., 1991). The alternate explanation, that Cx43 oligomerization occurs in a compartment proximal to the TGN but is inhibited by BFA-induced mixing of ER and Golgi contents, is unlikely since assembly is still blocked in the presence of nocodazole, an inhibitor of BFAmediated ER-Golgi fusion (Lippincott-Schwartz et al., 1990) (Figure 7). Further evidence in support of the TGN as the site of connexon formation is the fact that Cx43 is efficiently ‘assembled under conditions (20%) that lead to the accumulation of newly synthesized proteins in this compartment (Griffiths and Simons, 1986). Although highly suggestive, these data will have to be corroborated by additional studies (perhaps utilizing connexon-specific antibodies) before the precise s&cellular location of connexon oligomerization can be definitivelyestablished. We cannot, for example, currently rule out the formal possibility that connexon assembly occurs in a,pre-Golgi compartment distal to the site of the 15% block, such as that proposed by Bonatti et al. (1989) if transport of Cx43 into (or assembly within) such a compartment were blocked by BFA. In addition, it is conceivable that newly synthesized Cx43 is initially incorporated into a transient multimolecular complex within the ER that is unstable to Triton X-100 and is therefore undetectable in our assays. Solubilization of cells with other mild nonionic detergents (n-octyl glucoside or lauryl dimethylamineoxide) did not, however, result in increased recovery of Cx43containing oligomers (data not shown). Possible Role and Molecular Mechanism of Post-El3 Assembly of Connexons Why might connexins, unlike other integral plasma membrane proteins, normally be assembled after exit from the ER? One possible reason would be to prevent intracellular pairing of connexons and subsequent gap junctional plaque formation. The “gap” between the plasma membranes of two apposing cells in a vertebrate gap junctional plaque is -2-3 nm (C&par et al., 1988). If the distance between membrane bilayers on either side of the lumen of an ER or Golgi cisterna were less than or equal to this, then the extracytoplasmic (lumenal) domains of two connexons on opposite sides of the same ER or Golgi saccule might be able to interact and form a channel bridging the cisternal lumen. Such a structure might serve as a nucleation site for the formation of additional intracisternal channels, which could then cluster into gap junctional plaques inside the cell. Double bilayer gap junctions have, in fact, been reported within the ER (as well as on the plasma membrane) of BHKcell transfectants expressing very high levels of Cx32, another member of the connexin protein family (Kumar and Gilula, 1992; N. M. Kumar and N. B. Gilula, personal communication). Preliminary studies indicate that Cx43 is also assembled within the ER when overexpressed in transfected cells and that this premature assembly is a direct consequence of supraphysiological concentrations of newly synthesized connexin protein (L. S. M. and D. A. G., unpublished data). Should the associations between connexons in intracellular gap junctions be as stable as they appear to be in cell surface junctional

Cdl 1074

plaques (see Bennett et al., 1991), it would be highly unlikely that a connexin incorporated into such a structure could be transported to the cell surface. In normal CellS expressing endogenous levels of connexins, such nonproductive intracellular junction formation has not been reported and may be avoided by restricting connexon assembly to a compartment late in the secretory pathway. Once formed, connexons would be rapidly transported to the plasma membrane via TGNderived transport vesicles whose diameter (-50-100 nm; Griffiths et al., 1985) relative to that of a connexon (-9 nm; Caspar et al., 1988) and steep radius of curvature would provide a physical barrier to gap junction formation. Such a possibility would be in keeping with our experimental results supporting the TGN as the site of connexon assembly. The demonstration of a post-ER pathway for connexon oligomerization raises several fundamental questions concerning the currently accepted view of assembly of multisubunit integral plasma membrane proteins. First, how do connexin monomers in the ER escape destruction or (if incompletely or improperly folded) permanent aggregation, as is the commonly observed fate of unassembled subunits of other integral plasma membrane proteins (Rose and Doms, 1988; Huttley and Helenius, 1989; de Silva et al., 1990)? One possibility is that newly synthesized connexin molecules become transiently associated with an ER chaperone that protects them from such deleterious processes (Gething and Sambrook, 1992). Alternatively, ER retention and degradation may be mediated by specific protein sequences (Bonifacino and LippincottSchwartz, 1991; Nilsson et al., 1989; Wileman et al., 1990) that connexins lack. With a very few possible exceptions (Potter et al., 1985), assembly in the ER has previously been reported to be a prerequisite for the transport of endogenously expressed integral plasma membrane protein subunits through the secretory pathway(Hurtley and Helenius, 1989; Rose and Doms, 1988). How, then, are connexin monomers transported to the Golgi? It could be that in the absence of opposing factors (i.e., degradation, permanent aggregation, or an ER retention signal), transport from the ER to the Golgi is constitutive even for unassembled membrane protein subunits, as appears to be the case for monomeric proteins (Pfeffer and Rothman, 1987; Pelham, 1989) and for free subunits of some soluble proteins (Corless et al., 1987; Singh et al., 1990). Another possibility could be that an ER chaperone such as BiP might escort connexin molecules out of the ER, although presumably no further than the cis Golgi (Pelham, 1989). After arrival in the Golgi network, what triggers connexon assembly? The concentration of some proteins has been reported to be severalfold higher in the Golgi than in the Ek (Copeland et al., 1988; Quinn et al., 1984; Munro and Pelham, 1987). This is likely to be true for connexins as well, which appear to accumulate in the Golgi (relative to the ER) of numerous cell types both in culture (Musil et al.. 1990b: Berthoud et al.. 19921 and toossiblvl in vivo (Hendrix et al., 1992; Rahman et iI., 1993). If ti;? rate or extent (or both) of connexon assembly is sensitive to the local concentration of newly synthesized connexin protein, as is suggested by the studies of connexin-overexpressing

transfected cell lines mentioned above, then the increased density of connexin proteins in the Golgi may create conditions permissive for connexon oligomerization. This assembly might be assisted by Golgi chaperones as yet unknown or by physical differences between the ER and Golgi environments (e.g., intraluminal pH and ionic composition). Finally, is there a Golgi quality control system that prevents the transport of free connexin monomers to the cell surface? An intriguing possibility is that the postER oligomerization pathway that we have described for Cx43 is also utilized by other multisubunit plasma membrane proteins for which functional activation in early biosynthetic compartments may be undesirable. Experimental

Procedures

cell Culture The NRK, Sl60, and L929 cell lines were maintained as previously described (Musil et al., 1990b). Newly confluent 3-day-old 60 mm cultures were used for all experiments. The characterization and maintenance of the /d/F-2 and /d/G-42 temperature-sensitive CHO cell lines aredescribed by Fisher (1992). Both mutantcell lines, as wellascontrol wild-type CHO cells, were maintained in minimal essential medium (aMEM) supplemented with 6% fetal calf serum at 34OC.

Metabolic Labeling Cell Lyaates

of Cell Lines

and Preparation

of

[Y+methionine metabolic labeling of cells at 34°C-40DC was as previously described (Musil et al., 199Oa). For labeling of tissue culture cells at 15OC or 20°C, monolayers were incubated in reduced bicarbonate labeling medium (Earle’s minimal essential medium lacking methionine and containing 15 mM HEPES, 0.35 g/l bicarbonate, and 300 mg/l glutamine [pH 7.31) supplemented with [“Blmethionine (0.3 mCi per 60 mm dish of cells) and 5% dialyzed fetal calf serum and transferred to either a 15YI or 20°C ambient air (no CO2) incubator in a cold room. Temperatures were maintained within about 0.5% throughout the incubation. When desired, radiolabeled cells were chased in labeling medium supplemented with 0.5 mM methionine and 10% fetal calf serum. For experiments involving CCCP, the chase medium consisted of glucose-free minimal essential medium supplemented with 5% dialyzed fetal calf serum and 15 PM CCCP. Control cultures were chased in the absence of CCCP in the same medium supplemented with 4.5 g/l glucose. [=S]methionine-labeled tissue culture cells were scraped from the tissue culture dishes on ice with incubation buffer (0.14 M NaCI, 5.3 mM KCI, 0.35 mM Na2HPOc7H20, 0.35 mM KH*PO,, 0.6 mM MgSO,, 2.7 mM CaC12.2H20, and 20 mM HEPES [pH 7.51) (Musil and Goodenough, 1991). After centrifugation at 150 x g for 7 min, the cells were resuspended in incubation buffer (1 ml per 60 mm culture) supplemented with 0.5 mM diisopropyifluorophosphate, IO mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, and 200 PM leupeptin and then lysed by repeated passage through a 25gauge needle. Triton X-100 (20%) was added to a final concentration of 1%. After a 30 min incubation at 4OC, the lysates were subjected to centrifugation at 100,000 x g for 50 min at 4OC to separate Triton-insoluble (including gap junctional plaques) from Triton-soluble material (Musil and Goodenough, 1991). Unless otherwise noted, the supernatant fraction was then used for either chemical cross-linking or sucrose gradient fractionation (see below).

Sucrose

Gradient

Velocity

Sedlmentatlon

Analysis

Using the method described by Blount and Merlie (1966), -0.6 ml of the Triton-soluble supernatant prepared from [3SS]methionine-labeled cells as described above was fractionated on a linear gradient (4.0 ml total) of 5%-20% sucrose (w/v at 20°C) in incubation buffer and 0.1% Triton X-100. Centrifugation was performed in a Beckman SW60 rotor at 49,000 rpm for 12 hr (1.15 x IO” radiansVs) at 4OC, after which -4OO~lfractionswerecollectedfollowingpunctureof thetubebottom. The sucrose concentration in each fraction was measured usina a refractometer and was not corrected for the refractive index (1.3326,

Post-ER 1075

Assembly

of an Integral

Membrane

Protein

equivalent to - 1 .O% sucrose) of 0.1% Triton X-100 in incubation buffer in the absence of sucrose. Each fraction was then boiled in 0.6% SDS and immunoprecipitated with antiCx43 antibodies as described below. Samples were analyzed on 10% SDS-polyacrylamide gels, and the amount of Cx43 in each fraction was quantitated by laser densitometry after fluorography. No immunoprecipitable Cx43 pelleted to the bottom of the centrifuge tube under any of the conditions tested. Standard proteins (horseradish peroxidase, - 5s; catalase, 11.4s) were run on separate sucrose gradients to confirm that the 55 and 9S positions were as reported by Merlie and Blount (1988).

Chemical Cross-Linking ]“S]methionine-labeled cells were lysed with 1% Triton X-100 at 4’C and subjected to centrifugation at 106,ooO x g as described above. The Triton-soluble supernatant of this centrifugation was diluted with 2 vol of incubation buffer (see above) and then incubated on ice with either DSP (from a fresh 7.5 mglml stock solution in dimethyl sulfoxide [DMSO]) (Pierce Chemical Company), EGS (from a 46 mg/ml stock in DMSO), or an equal volume of DMSO. After 30 min at 4OC, the cross-linking reaction was quenched by addition of 20 fulml of a 1 M glycine stock (pH 9.2) and the samples were incubated on ice for an additional 30 min. Finally, 10 pllml of 1 M glycine (pH 7.2) was added to lower the pH, and the samples were boiled in SDS (final concentration, 0.6%) for 3 min. Cx43 was immunoprecipitated from the samples as described below and analyzed on 4%-15% polyacrylamide gradient gels in the absence of reducing agent unless otherwise noted. In all cross-linking experiments presented except those in Figure 2, the stacking gel is also shown. High molecular mass standards included ap-macroglobulin (unreduced, 360 kd; reduced, 180 kd). Attempts to cross-link intact NRK cells or NRK cell membranes in the absence of Triton X-100 led to the loss of both 40 kd and 260 kd forms of Cx43 and their incorporation into very large heterogeneous aggregates (data not shown). This is probably due both to inter-connexon cross-linking in the tightly packed gap junctional plaques and to nonspecific crosslinking of highly concentrated but unrelated neighboring proteins in the membrane plane (Ji, 1979).

Expression

of Cx43

in Xenopus

Oocytes

Stage VI Xenopus oocytes were collected, defolliculated, and microinjetted with in vitro transcribed rat Cx43 complementary RNA (cRNA) (- 10 ng/40 nl per oocyte) as detailed by Swenson et al. (1989). After an overnight incubation at 15OC in Ca’containing modified Barth’s saline, the oocytes were metabolically labeled by addition of [35S]methionine (0.1 mCillO0 ul) to fresh modified Barth’s saline and incubated for 4 hr at 15OC. After labeling, the oocytes were carefully rinsed in incubation buffer (see above) and lysed at 4°C in 250 ul per oocyte of incubation buffer supplemented with 0.5 mM diisopropylfluorophosphate, 10 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 200 uM leupeptin, and 1 .O% Triton X-100. The lysates were then subjected to centrifugation for 5 min at 10,060 x g at 4OC to pellet yolk granules, and the supernatant was incubated for 30 min at 4OC prior to a second centrifugation at 106,000 x g for 50 min at 4OC. The supernatanl of this spin was then subjected to either chemical crosslinking or to sucrose gradient fractionation exactly as described for tissue culture cell lysates (see above).

Immunopreclpltatlon

of Cx43

After sucrose gradient fractionation or cross-linking (see above), samples boiled in 0.6% SDS were diluted with 4 vol of immunoprecipitation buffer(O.1 M NaCI, 0.02 M sodium borate, 15mM EDTA, 15mM EGTA, 0.02% sodium aside, 10 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride [pH 6.51) supplemented with 0.5% Triton X-l 00 and immunoprecipitated according to the procedure of Musil et al. (1990a). The antiCx43 antibodies used throughout this study were affinity purified from a rabbit antiserum generated against a Cx43epecific peptide encoding amino acids 252-271 of rat heart Cx43 as previously described (Musil et al., 1990b). Control immunoprecipitations were conducted with either the preimmune serum from this rabbit or with affinitypurified rabbit antibodies directed against residues 114-133 of an irrelevant connexin, Xenopus Cx36. Both preparations gave similar results.

lmmunofluorescent

Localization

of Cx43

Cell cultures grown on untreated 35 mm tissue culture dishes were fixed in 1% formaldehyde, permeabilized with 0.2% Triton X-100, and stained with affinity-purified antiCx43(252-271) antibodies followed by rhodamine-labeled goat anti-rabbit immunoglobulin G as in Musil et al. (1990b).

Acknowledgments Experiments with the /d/F and /d/G cell lines were conducted in the laboratory of Monty Krieger (Massachusetts Institute of Technology), and we are very grateful to him for providing these cells and especially to Dr. Abby Fisher for her expertise and advice in their use. We also thank Tom White and Roberto Bruuone for their invaluable help with the Xenopus oocyte experiments and Tom Wileman for his gift of the BFA (originally obtained from Dr. Jennifer Lippincott-Schwartz) used in preliminary experiments. This work was supported in part by grants GM-18974 and EY-02430 to D. A. G. from the National Institutes of Health. L. S. M. is a fellow of the Medical Foundation of Boston. Received

May 14, 1993;

revised

July 14, 1993

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