(Received for publication, June 7, 1995; and in revised form, August 9, 1995)
From the
We investigated the degradation of the gap junction protein
connexin43 in E36 Chinese hamster ovary cells and rat
cardiomyocyte-derived BWEM cells. Treatment of E36 cells with the
lysosomotropic amine, primaquine, for 16 h doubled the amount of
connexin43 detected by immunoblotting and modestly increased the
half-life of connexin43 in pulse-chase studies, suggesting that the
lysosome played a minor role in connexin43 proteolysis. In contrast,
treatment with the proteasomal inhibitor N-acetyl-L-leucyl-L-leucinyl-norleucinal led
to a 6-fold accumulation of connexin43 and increased the half-life of
connexin43 to 9 h. The role of ubiquitin in connexin43 degradation
was examined in an E36-derived mutant, ts20, which contains a
thermolabile ubiquitin-activating enzyme, E1. E36 and ts20 cells grown
at the permissive temperature contained similar amounts of connexin43
detectable by immunoblotting. Heat treatment dramatically reduced the
amount of connexin43 detected in E36 cells, while connexin43 levels in
heat-treated ts20 cells did not change. E36 cells that were
heat-treated in the presence of N-acetyl-L-leucyl-L-leucinyl-norleucinal did
not lose their connexin43. Pulse-chase experiments showed the
reversibility of the block to connexin43 degradation in ts20 cells that
were returned to the permissive temperature. Finally, sequential
immunoprecipitation using anti-connexin43 and anti-ubiquitin antibodies
demonstrated polyubiquitination of connexin43. These results indicate
that ubiquitin-mediated proteasomal proteolysis may be the major
mechanism of degradation of connexin43.
Gap junctions are plasma membrane structures that contain groups of channels that allow the passage of ions and small molecules between adjacent cells(1) . While their microscopic appearance suggests that gap junctions might be stable structures, in fact, studies have suggested that they are quite dynamic. The turnover and degradation of gap junctions may have profound consequences for intercellular interactions in several physiological or pathological situations. Examples are as follows: hepatocyte gap junctions are rapidly degraded, and cells uncouple after partial hepatectomy followed by gap junction resynthesis associated with liver regeneration(2) ; ventricular myocyte interconnections at gap junctions are remodeled and redistributed in the zones bordering healed myocardial infarcts in a manner that may predispose to reentrant arrhythmias(3) , and gap junctions in uterine myometrium are synthesized rapidly at term and degraded rapidly after delivery leading to uncoupling of these smooth muscle cells(4) .
The turnover of gap junctions and their
constituent proteins also appears to be rapid in normal adult organs
and in cultured cells. Fallon and Goodenough (5) determined
that hepatic gap junctions have a half-life of about 5 h in
vivo. Further turnover studies were facilitated by the molecular
cloning of the subunit gap junction proteins (connexins) and the
development of specific antibodies. Use of these antibodies for
immunoprecipitation of radioactively labeled connexins in pulse-chase
experiments has shown that connexin43 (Cx43), ()connexin26,
connexin32, and connexin45 have half-lives of 2-3 h in cultured
cells(6, 7, 8, 9) . Compared with
many other membrane proteins, gap junction proteins turn over rather
rapidly. These findings suggest that even under normal conditions,
synthesis and degradation of gap junctional channels are very dynamic
processes and may provide major mechanisms for the regulation of
intercellular coupling and potential remodeling of cellular
connections.
The major pathways of protein degradation include lysosomal proteolysis, ubiquitin-dependent lysosomal proteolysis (autophagy), ubiquitin-dependent proteasomal proteolysis, and ubiquitin-independent proteasomal proteolysis. Lysosomes are predominantly involved in the degradation of internalized extracellular materials and receptors. Ubiquitination may regulate the proteolysis of many short-lived and abnormal cellular proteins(10) . The proteasome may degrade proteins in a non-ubiquitin-dependent manner (11, 12, 13) , and ubiquitin is involved in autophagy(14) .
To elucidate mechanisms of gap junction degradation, we characterized Cx43 turnover and degradation in E36 Chinese hamster ovary cells, in rat cardiac myocyte-derived BWEM cells, and in a mutant of the E36 cells (ts20). The ts20 cells have a temperature-sensitive defect in the ubiquitin-activating protein, E1, and fail to degrade short-lived or abnormal proteins at the restrictive temperature(10) .
To determine possible mechanisms of Cx43 degradation, we treated E36 or BWEM cells with several different protease inhibitors and determined Cx43 levels by immunoblotting and densitometry. (Similar results were seen with both cell lines; those obtained using E36 cells are shown in Fig. 1). Cells were treated for 16 h with either the lysosomotropic amine, primaquine (200 µM), or two inhibitors of neutral cysteine proteases, ALLN (20 µM) and ALLM (20 µM). ALLN and ALLM are similar and can both inhibit calpain and cathepsin D; however, at the concentrations used, only ALLN inhibits proteasomal degradation(13) . The electrophoretic mobilities of Cx43 were similar in control and treated samples. The amount of Cx43 was modestly increased (2-fold) in E36 cells treated with primaquine (200 µM) as compared with control cultures (Fig. 1, lanes 1 and 2). E36 cells treated with ALLM contained similar amounts of Cx43 to control cultures (1.2 times as much by densitometry) (Fig. 1, lanes 1 and 4). In contrast, E36 cells treated with ALLN contained 6 times as much Cx43 as control cultures (Fig. 1, lane 3). These data suggested that proteasomal degradation might be the major route of Cx43 proteolysis.
Figure 1: Immunoblot analysis of E36 cells treated with various reagents. Cultures of E36 cells (60-80% confluent) were not treated (lane 1) or were treated with 200 µM primaquine (lane 2), 20 µM ALLN (lane 3), or 20 µM ALLM (lane 4). These cells were harvested, and 5-µg aliquots of each were resolved on a 10% polyacrylamide gel and transferred to Immobilon-P. These membranes were probed with the anti-Cx43 monoclonal antibody and detected by chemiluminescence.
To further test
mechanisms of Cx43 degradation, cells were labeled with
[S]methionine for 2 h and then were chased with
fresh medium containing 2 mM methionine in the presence or
absence of protease inhibitors. The cells were harvested and Cx43 was
immunoprecipitated and analyzed by electrophoresis and fluorography. Fig. 2shows the results of a representative pulse-chase
experiment in control E36 cells or primaquine-treated E36 cells. Cx43
was detected as a doublet at 42 and 44 kDa, and the amount of
[
S]methionine-labeled Cx43 diminished throughout
the labeling period in both control and primaquine-treated cultures.
Densitometric quantitation of these immunoprecipitations showed that
the half-life of Cx43 was 2.5 h in the control and 3 h in the
primaquine-treated cultures. While the turnover of Cx43 differed only
mildly, examination of total
[
S]methionine-labeled proteins by SDS-PAGE and
fluorography confirmed that the primaquine had inhibited the
degradation of many other cellular proteins (data not shown).
Figure 2:
Pulse-chase analysis of Cx43 turnover in
normal and primaquine-treated cells. Cultures were labeled for 2 h with
[S]methionine (100 µCi/ml) and chased for
0-4 h in normal media with or without 200 µM primaquine. All cultures were immunoprecipitated with anti-Cx43
antisera and then analyzed by SDS-PAGE and fluorography (inset). The fluorographs were analyzed by densitometry, and
the relative amounts of Cx43 found at each time point are shown on the
graph. The solid line represents data from control cultures,
while results from primaquine treated cultures are shown as a dashed line.
In contrast, the turnover of Cx43 was significantly prolonged by the proteasomal inhibitor ALLN in both cell lines. The amount of Cx43 immunoprecipitated from E36 cells chased for 3-6 h in the presence of 20 µM ALLN was not significantly different from that at the beginning of the chase period (data not shown). Experiments with the chase period extended to 24 h showed that the Cx43 protein had a half-life of 9 h in BWEM cells (Fig. 3). Taken together, these immunoblot and pulse-chase experiments suggested that there was limited involvement of lysosomal proteolysis but a major role of the proteasome in Cx43 degradation in these cells.
Figure 3:
Pulse-chase analysis of Cx43 turnover in
normal and ALLN treated cells. Cultures of BWEM cells were labeled for
2 h with [S]methionine (100 µCi/ml) and
chased for 0-24 h in normal media, with or without 20 µM ALLN, immunoprecipitated with anti-Cx43 antisera and then analyzed
by SDS-PAGE and fluorography (inset). The fluorographs were
analyzed by densitometry, and the relative amounts of Cx43 found at
each time point are depicted graphically. Data from control cultures
are represented by the solid line, while those from
ALLN-treated cultures are shown as a dashed
line.
The role of the ubiquitin-proteasome system in Cx43 degradation was examined in heat-treated ts20 and E36 cells. Heat treatment of many cells, including the E36 cells, leads to a stress-induced degradation of many cellular proteins. But heat treatment (44 °C for 1 h followed by 2 h at 39.5 °C) of ts20 cells inactivates the thermolabile E1 enzyme and thereby inactivates the ubiquitin-conjugation system(10) , as we confirmed by an ATP-dependent ubiquitination assay in lysates of cultures of both E36 and ts20 cells (data not shown). Cellular lysates of heat-treated E36 and ts20 cells were analyzed by immunoblotting with a monoclonal antibody directed against Cx43 (Fig. 4). Untreated ts20 and E36 cells contained similar amounts of Cx43 detected as a major band of 42 kDa (Fig. 4, lanes 1 and 2). Much less Cx43 was detected in heat-treated E36 cells (Fig. 4, lane 4). Inactivation of ubiquitination apparently prevented the loss of Cx43 in ts20 cells, since heat-treated ts20 cells retained a substantial fraction of the original amount of Cx43 (Fig. 4, lane 3). The loss of Cx43 was blocked in E36 cells that were heat-treated in the presence of ALLN (Fig. 4, lane 5), but not ALLM (Fig. 4, lane 6). These results indicate that the proteolysis of Cx43 in heat-treated E36 cells is dependent on ubiquitination and the proteasome. In addition to these differences, the mobility of some of the Cx43 was reduced in heat-treated cells, suggesting changes in phosphorylation may accompany heat-treatment.
Figure 4: Immunoblot analysis of Cx43 content of untreated (lanes 1 and 2) or heat-treated (lanes 3-6) ts20 (lanes 1 and 3) or E36 (lanes 2, 4, 5, and 6) cells. Parallel cultures of E36 cells were heat-treated in the presence of 20 µM ALLN (lane 5) or 20 µM ALLM (lane 6). Cultures of ts20 or E36 cells were heat-treated (44 °C for 1 h followed by 2 h at 39.5 °C) or maintained at 30.5 °C, followed by harvesting preparation of cell lysates and analysis by immunoblotting with a monoclonal antibody directed against Cx43.
To examine the cellular location of Cx43 in these cells, cultures of E36 and ts20 cells were heat-treated, fixed, and permeabilized, and Cx43 was detected by immunofluorescence (Fig. 5). At permissive temperatures (30.5 °C), moderate amounts of Cx43 staining were found at appositional membranes between either ts20 or E36 cells (Fig. 5, A and C) as expected for a gap junctional protein. Heat treatment led to some reduction of the amount of Cx43 detected in the E36 cells; this residual staining was still apparent at the cell membrane (Fig. 5B). After heat treatment of the ts20 cells, the distribution of immunoreactive Cx43 appeared substantially different; while some of the staining appeared to be at appositional membranes, substantial cytoplasmic staining was also detected (Fig. 5D). This staining appeared to be diffuse within the cytoplasm.
Figure 5: Immunofluorescence analysis of Cx43 in untreated (A and C) or heat-treated (B and D) ts20 (A and B) and E36 (C and D) cells. Cultures of ts20 or E36 cells were heat-treated (as in Fig. 3) or maintained at 30.5 °C. Cultures were fixed and permeabilized and stained with a monoclonal antibody directed against Cx43 as detected by indirect immunofluorescence (bar = 50 µm).
We performed further pulse-chase
experiments to confirm the reversibility of the accumulation of Cx43
induced by heat treatment, as would be predicted by the reversibility
of E1 inactivation. Cultures of ts20 cells were incubated at 44 °C
for 1 h, followed by incubation in the presence of
[S]methionine in methionine-depleted media for 2
h at 39.5 °C. One culture was then harvested for
immunoprecipitation, while parallel cultures were chased in normal
media for 3 h at 39.5 or at 30.5 °C. After the chase period, cells
were harvested and lysed in RIPA buffer, and Cx43 was
immunoprecipitated and analyzed by SDS-PAGE and fluorography. The cells
chased at 39.5 °C (Fig. 6, lane 3) contained
comparable amounts of Cx43 (42 and 44 kDa bands) to the sample from the
start of the chase (Fig. 6, lane 1), while the cells
chased at 30.5 °C contained only 10% as much Cx43 (44 kDa band
only) confirming that degradation of Cx43 was restored (Fig. 6, lane 2).
Figure 6:
Reversibility of heat-induced block to
Cx43 degradation in ts20 cells. Cultures of ts20 cells were incubated
at 44 °C for 1 h followed by 2 h at 39 °C in the presence of
[S]methionine (100 µCi/ml) in
methionine-depleted media, after which Cx43 was immunoprecipitated from
one culture (lane 1). Cultures were chased in normal media for
3 h at either 30.5 °C (lane 2) or 39.5 °C (lane
3), and then Cx43 was immunoprecipitated. The immunoprecipitated
Cx43 was analyzed by SDS-PAGE and fluorography.
In order to detect evanescent polyubiquitinated
forms of Cx43, we performed immunoprecipitation experiments using E36
cells that were metabolically labeled for 16 h with
[S]methionine in the presence of ALLN to inhibit
proteasomal activity. These cells were harvested in RIPA buffer and
sequentially immunoprecipitated with antibodies directed against Cx43
followed by an anti-ubiquitin monoclonal antibody. The material
immunoprecipitated by anti-Cx43 antibodies was detected as two major
bands of 42 and 44 kDa after an overnight exposure (Fig. 7, lane 1). In contrast, subsequent immunoprecipitation with the
anti-ubiquitin antibody precipitated a number of bands between 44 and
70 kDa (Fig. 7, lane 2) detected only after a much
longer exposure (3 weeks). These bands were not seen when the
immunoprecipitation was performed in the presence of an excess of
competing ubiquitin (Fig. 7, lane 3). These results
appeared similar to the ladders of ubiquitinated polypeptides detected
with other substrates of the ubiquitin/proteasomal
apparatus(22) .
Figure 7:
Immunoprecipitation of Cx43-ubiquitin
conjugates. E36 cells were metabolically labeled with
[S]methionine for 16 h in the presence of ALLN
(20 µM) and harvested for immunoprecipitation. Lysates
were immunoprecipitated with a rabbit antiserum directed against Cx43 (lane 1); parallel cultures were immunoprecipitated with a
rabbit antiserum directed against Cx43 followed by reprecipitation with
a monoclonal antibody against ubiquitin (lane 2) or
reprecipitation with an anti-ubiquitin monoclonal antibody in the
presence of competing ubiquitin (100 µg/ml) (lane 3).
The major conclusion of this study is that the degradation of
Cx43 is dependent upon ubiquitin and the proteasome. This conclusion is
supported by several observations of E36, BWEM, and ts20 cells. First,
Cx43 accumulated in E36 or BWEM cells treated with the proteasomal
inhibitor ALLN, due to decreased turnover of the protein. Second, while
in the normal E36 cells heat treatment induced degradation of Cx43, in
heat-treated ts20 cells containing a thermolabile ubiquitination
pathway, Cx43 remained abundant as assessed by immunoblotting,
immunofluorescence, and immunoprecipitation. Third, the heat-induced
degradation of Cx43 was blocked by the proteasomal inhibitor ALLN but
not by equal concentrations of the related protease inhibitor ALLM.
While calpains are inhibited by similar concentrations of both of these
neutral cysteine protease inhibitors, the proteasome is inhibited much
more effectively by ALLN than by ALLM(13) . Fourth, the blocked
Cx43 degradation in heat-treated ts20 cells was reversible as assessed
by pulse-chase experiments in which returning heat-treated ts20 cells
to 30.5 °C restored the proteolysis of Cx43. Fifth,
polyubiquitinated Cx43 conjugates were isolated from ALLN-treated E36
cells. These could serve as substrates for the proteasome. The rather
long exposure time required for their detection and the necessity for
inclusion of ALLN, suggested that the ubiquitinated forms of Cx43 may
constitute only a small fraction of the total Cx43 pool and may have
only a very short lifespan. Sixth, in ts20 cells stably transfected
with a cDNA encoding the human E1 enzyme(18) , we have found
that Cx43 does not accumulate after heat-treatment, ()confirming that the block to degradation in the ts20 cells
is due to the lability of this enzyme.
Our data implicate ubiquitination and the proteasome in the degradation of a very hydrophobic plasma membrane protein, the gap junction protein Cx43. While ubiquitin modification has been implicated in a variety of cellular processes including regulating gene expression, cell cycle and division, the cellular stress response, modification of cell surface receptors, DNA repair, import of proteins into mitochondria, uptake of precursors into neurons, and the biogenesis of ribosomes, mitochondria, and peroxisomes, its exact role in most of these processes is unclear (23) . In contrast, the role of the ubiquitin modification in the proteasomal pathway of protein degradation has been extensively characterized, and polyubiquitination is a signal for the proteasome to recognize proteins marked for degradation(23) . Numerous cytoplasmic and nuclear proteins involved in signal transduction and cellular replication are substrates of the ubiquitin-proteasome pathway including N-myc, c-myc, c-fos, and p53 (24) . Several plasma membrane proteins, including the c-kit protein(25) , Ste6(26), and the T-cell antigen receptor(27) , are modified by polyubiquitin moieties. Mori et al.(28) have shown that proteolysis of another plasma membrane protein, the platelet-derived growth factor receptor, is dependent upon polyubiquitination. Taken together, these studies suggest that ubiquitin plays a role in degradation of proteins from many different compartments of the cell.
Our findings imply that polyubiquitination is the signal for Cx43 degradation, but the structural determinants of Cx43 ubiquitination are currently unknown. Cx43 ubiquitination is unlikely to be governed by the N-end rule(30) ; it contains ``stabilizing'' residues as its first two amino acids (methionine and glycine), and N-terminal microsequencing (30) indicates that the amino-terminal residue in Cx43 isolated from rat tissues is glycine. Cx43 might possibly be recognized by a similar ubiquitin protein ligase (E3) and ubiquitin-carrier protein (E2) to those needed to catalyze the proteolysis of another non-N-end rule protein, p53, which is a well known substrate of the ubiquitin proteasome pathway(31) . Some proteins that have short half-lives contain a sequence rich in proline, glutamic acid, serine, and threonine residues called a PEST sequence(32) . As discussed by Laird et al.(8) , potential PEST sequences are present in the Cx43 protein. These sequences may correlate with rapid degradation of PEST box bearing proteins and have recently been shown to play a role in the ubiquitination and degradation of Cln3 cyclin by the ubiquitin-conjugating enzyme Cdc34(33) .
We believe that the ubiquitin proteasome pathway may also be a major mechanism for the proteolysis of gap junctions and connexins in other systems. Our data demonstrate involvement of ubiquitination and the proteasome in the normal turnover of Cx43 in two different cell lines and in the accelerated degradation induced by heat stress. Studies of the proteolysis of connexin32 have suggested that this protein may be digested by calpains(34) . A Lewis lung carcinoma cell line major histocompatibility complex class I antigen contained an octapeptide differing only at a single position from a portion of mouse connexin37 (35) . These cells contained and expressed both mutant and wild-type connexin37 genes, but only the mutant connexin37 peptide was associated with the major histocompatibility complex class I complex(35) . Thus, this mutant connexin37 protein was proteolyzed by the ubiquitin proteasome pathway(13) . Immunohistochemical studies of cultured cardiac myocytes and heart tissue using anti-ubiquitin conjugate antibodies indicated the presence of ubiquitin conjugates in the intercalated disc(36) , further suggesting that components of intercellular junctions may be substrates of the proteasome.
Because we observed a 2-fold Cx43 accumulation and a minor prolongation of its half-life in the presence of primaquine, we cannot exclude any role for the endosomal/lysosomal pathway in Cx43 degradation. But, in contrast to our observations, the half-life of P-selectin, a membrane protein that is degraded in the lysosome, is extended significantly by lysosomotropic amines(37) . Therefore, we have concluded that the lysosome has only a minor role in Cx43 proteolysis. While we observed accumulation of Cx43 within the cytoplasm of heat-shocked ts20 cells, this staining appeared diffuse, not vesicular. In contrast, several previous morphological studies reported vesicular structures containing morphologically identifiable gap junctions called annular gap junctions; such studies suggested that gap junctions were internalized by endocytosis. Treatments that lead to a loss of morphologically identifiable gap junctions, such as tissue dissociation, ischemia, or anoxia in the liver increase the abundance of annular gap junctions(2) . In some tissue culture systems, these vesicles appear to be clathrin coated and associated with actin(38) , phagolysosomes(39) , or multi-vesicular ``complex structures''(40) . Further biochemical analyses in such systems will be required to test the generality of our conclusions.