From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343-355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.
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INTRODUCTION |
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Translocation of membrane proteins is a complex process. It involves recognition and targeting of the proteins to the endoplasmic reticulum (ER)1 membrane, their insertion into the lipid bilayer with their correct transmembrane (TM) orientation without interrupting the permeability barrier of the membrane bilayer, and finally, their maturation into functional proteins that includes co- and post-translational processes such as signal peptidase (SPase) cleavage of ER target signal sequences, folding, disulfide bridge formation, glycosylation, and other post-translational modifications, and eventually oligomerization. A large portion of the current knowledge on the synthesis and translocation of secretory and membrane proteins has been obtained by synthesizing these proteins in translation competent cell lysates supplemented with ER-derived membrane vesicles (microsomes) (1). Since the lysate system has been found in many cases to accurately reproduce the steps involved in translation, translocation, and co- and post-translational modifications, it has become a standard assay system to study these processes. Many secretory and predominantly single spanning TM proteins have been investigated using this assay system, and a large body of information on the mechanisms involved in the membrane translocation of these protein types has been accumulated (reviewed in Refs. 2-4). However, little is known about the synthesis of polytopic membrane proteins that traverse the membrane bilayer more than once. This holds true in particular for membrane channel proteins that bear charged amino acid residues within their TM domains to allow the formation of a hydrophilic pore within the membrane bilayer. Experimental evidence indicates that secretory, as well as TM proteins use the same translocation machinery, and that many of the steps that are involved in the biogenesis of both protein types are closely related (3, 5, 6). However, several recent discoveries indicate that the current understanding of membrane protein translocation is quite incomplete, and it may be much more versatile than previously expected (reviewed in Ref. 4).
We used connexin (Cx) polypeptides as model proteins to study the steps involved in the synthesis of channel forming membrane proteins. Cxs are the proteins that form gap junctions, TM channels providing direct cell-to-cell communication. They are nonglycosylated, polytopic plasma membrane (PM) proteins (class III membrane proteins) that traverse the membrane bilayer four times with NH2 and COOH termini located in the cytoplasm (Ncyt-Ccyt orientation). This TM topology was suggested by hydropathy analyses of the primary amino acid sequences and is supported by many other topological analyses (cited in Ref. 7). More than a dozen different Cxs have been cloned and sequenced from rodents, and all represent structurally conserved members of a multigene family that mainly differ in the length of their COOH-terminal domains. To form a gap junction channel, 6 Cxs oligomerize into a ring-shaped structure with a central hydrophilic pore, termed connexon. Two connexons, each provided by two neighboring cells then pair in the adjacent PMs to form the transcellular, double-membrane channel (reviewed in Refs. 8 and 9).
We found that the translation of Cx polypeptides in standard cell-free translation/translocation systems supplemented with ER-derived microsomes resulted in a complete, but inappropriate proteolytic processing that affected all Cx polypeptides upon their membrane integration (reported in this study and in Ref. 7). Functional Cxs organized in gap junction plaques at the PM are not processed in this manner (10, 11). This difference in Cx membrane integration in vitro and in vivo suggested that the membrane integration process of Cxs somehow differs from the membrane integration of previously studied membrane proteins and may require additional factors which are absent in the cell-free translation system. Further analysis showed that a comparable cleavage product was also detectable in the ER membranes of different cell types that overexpress Cxs. There, the amount of cleaved Cx polypeptides correlated with the expression level (7). The cleavage reaction clipped off an NH2-terminal portion of the Cx polypeptides, and appeared to be similar to the cleavage of target signal peptides (SP) from pre-proteins synthesized on the ER membrane. Further characterization indeed suggested a cleavage by SPase (12), the enzyme that normally cleaves NH2-terminal ER target SPs from secretory and certain TM pre-proteins oriented with their NH2 terminus located in the lumen of the ER. Cxs, however, are oriented with their NH2 terminus facing the cytoplasm, and no NH2-terminal SP is encoded within their NH2 terminus. Instead, internal hydrophobic signal anchor (SA) sequences have been characterized in Ncyt-oriented membrane proteins, such as the Cxs, that function in their membrane targeting and anchoring (reviewed in Refs. 2-4). Consequently, Cxs should not be cleaved by this enzyme upon their membrane integration.
Here we report the TM topology of Cxs generated in ER membranes, and that a modified TM orientation is not causing this inappropriate cleavage. Furthermore, we performed an extensive characterization of the cleavage site, reaction, and conditions that are associated with this proteolytic processing. The results indicated that the SA sequence of Cxs was improperly recognized and positioned within the ER translocon when Cxs were translocated in vitro, and that this mis-positioning allowed SPase to access the cleavage sites in the Cx sequences. The results provide direct evidence that additional, unknown cellular factors are involved in the successful membrane integration of Cx polypeptides in vivo that are absent or not functional in standard cell-free translation/translocation systems.
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EXPERIMENTAL PROCEDURES |
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cDNA Clones, Chimeras, and Mutagenesis of Cx
Polypeptides--
For the in vitro transcription of
efficiently translated synthetic Cx RNAs, rat 1 (Cx43),
and human
1 (Cx32) cDNAs were cloned into the
transcription vector pSP64T (13) as described in Falk et al.
(7).
In Vitro Transcription, Translation, and Membrane Translocation
Assay--
All plasmids used for in vitro transcription
were linearized, phenol/chloroform extracted, and ethanol precipitated
prior to RNA synthesis. Transcription reactions were performed using T3, T7, or SP6 RNA polymerase (Promega Biotech, Madison, WI). The
quality and amount of synthesized RNA was determined by analyzing aliquots on agarose gels. Synthetic RNAs were either used immediately or stored in aliquots at 70 °C.
Pretreatment of Microsomal Vesicles-- Microsomal vesicles were depleted of their lumenal proteins by perforating them at high pH as described (19), except that CAPS (pKa 10.4) was replaced with CHES (pKa 9.3). 100-µl microsome aliquots were diluted 10-fold with either 50 mM (final concentration) HEPES buffer (pKa 7.55), pH 7.4, or 50 mM (final concentration) CHES buffer, pH 9.5 and pH 10.2, and incubated on ice for 30 min. Microsomes were neutralized and collected as described (19), and stored on ice until used. Depletion of their soluble, lumenal proteins was monitored by analyzing the protein content in the supernatants on Coomassie Blue-stained SDS-PAGE gels. To ensure protease inhibitor access to the lumen, microsomes were perforated as described above and preloaded with N-ethylmaleimide (NEM; 0.25 and 1 mM final concentration), iodoacetamide (1 mM final concentration), EDTA (1 mM final concentration), EGTA (2 mM final concentration), or diisopropyl fluorophosphate (2 mM final concentration) (Sigma) by adding these inhibitors to the perforation buffers.
Radiosequencing of 1 and
1
Cx--
For radiosequencing, Cxs were labeled with
L-[2,3,4,5,6-3H]phenylalanine (37 MBq/ml,
Amersham Corp.). A complete amino acid mixture (1 mM final
concentration) lacking phenylalanine (Phe) was prepared from amino acid
calibration markers (Sigma). A standard translation reaction (60 µl)
consisted of 30 µl of reticulocyte lysate, 3 µl of 1 mM
amino acid mixture without Phe, 15 µl of [3H]Phe, 1-3
µg of synthetic RNA, and 6-8 equivalents of microsomes. After
translation the reaction was loaded on SDS-PAGE gels, and the areas
containing the
1 or
1 Cx polypeptides
were excised, cut into smaller pieces, and placed into the chamber of
an ELUTRAP elution device (Schleicher & Schuell, Keene, NH). The
elution buffer (0.1% SDS, 20 mM
NH4HCO3, 1 mM thioglycolic acid,
0.1% dithiothreitol) was continuously mixed by pumping the buffer from the negative pole to the positive pole with a peristaltic pump (Bio-Rad). Proteins were eluted overnight at 100 V. The eluate (~1
ml/chamber) was dried and SDS removed by ion-pair extraction with
unhydrous acetone/triethylamine/acetic acid/water (85:5:5:5). Cxs were
then solubilized in 0.1% SDS solution and protein corresponding to
~100,000-200,000 cpm were applied to a Polybrene-treated glass fiber
filter for 20-25 cycles of amino acid sequence analysis in an Applied
Biosystems 470A gas-phase sequenator (Foster City, CA). Individual
cycles were collected before on-line high performance liquid
chromatography analysis. 50-µl aliquots were mixed with scintillation
mixture (Bio-Safe II, Research Products International Corp., Mount
Prospect, IL) and analyzed by liquid scintillation counting.
Radioactive peak activities were aligned with phenylalanine residues in
the Cx sequence to determine the exact position of the cleavage
site.
Gel Electrophoresis, Autoradiography, and
Densitometry--
Translation products were analyzed on 10 and 12.5%
SDS Laemmli gels (acrylamide/bisacrylamide ratio, 29:1). Samples were
solubilized in SDS sample buffer containing 3% SDS, 5%
-mercaptoethanol, and analyzed without heating to prevent
aggregation of Cx polypeptides. Following electrophoresis, gels were
soaked for 10 min in 1 M sodium salicylate (Sigma) to
enhance 35S and 3H-autoradiography, dried, and
exposed to Kodak X-AR film at
70 °C using an intensifying screen.
To determine cleavage efficiency of the SPase cleavage site mutants,
autoradiographs were scanned densitometrically using an AlphaImager
2000 Digital Imaging and Analysis System (Alpha Innotech Corp., San
Leandro, CA). Average cleavage efficiencies of the mutants were
expressed in percent compared with the cleavage efficiency of wild type
1 Cx.
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RESULTS |
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Cell-free Expression of Cxs--
For the analysis of the Cx
biogenesis described in this study, the two most abundant Cx isotypes,
heart Cx 1 (Cx43) and liver Cx
1 (Cx32),
were used. As we reported previously, Cx polypeptides can be expressed
efficiently as full-length proteins in reticulocyte lysates or wheat
germ extracts in the absence of microsomes. Synthetic Cx RNAs produced
translation products that migrated with an electrophoretic mobility on
SDS-PAGE gels that corresponded to their predicted molecular weights
(Fig. 1, lane 1, shown for
1 Cx; see also Ref. 7). However, translation in the
presence of pancreatic microsomes resulted in the generation of cleaved
Cxs with a faster mobility on SDS-PAGE gels (Fig. 1, lane 2)
that were retained in the microsomal membrane fraction after pelleting
the microsomes through a 0.5 M sucrose cushion (Fig. 1,
lanes 3 and 4). All Cx isotypes examined thus far
were aberrantly processed in this manner (7). Full-length Cx
polypeptides also generated in the presence of microsomes (Fig. 1,
lane 2) were not membrane associated and stayed in the
supernatant fraction (Fig. 1, lane 3), indicating that they
were synthesized by free ribosomes that failed to bind to the
membranes. Other secretory and transmembrane proteins, such as
prolactin (PL), MP26, and synaptophysin (Syn), translated in parallel
reactions in the presence of microsomes gave results as reported
previously, namely SP cleavage of pre-prolactin (20), membrane
integration of MP26 without SP cleavage or glycosylation (21), and
membrane integration and N-glycosylation of Syn (22),
respectively (Fig. 1, lanes 5-10).
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The TM Topology of Cxs Integrated into ER Membranes Was Identical
to Their Topology in the PM--
The TM topology of Cxs integrated
into microsomes was determined by N-glycosylation site
tagging. N-Glycosylation consensus sequences were introduced
into the extracellular loops (E1 and E2), and into the intracellular
loop (I-loop) described to be located on both sides of the PM (Fig.
2A). While the
1 mutants with the consensus sites in the extracellular
loop E1 (designated
1E1K48N and
1E1L56S),
or E2 (designated
1E2L165N/K167S) were efficiently
glycosylated resulting in a reduced mobility on SDS-PAGE gels (Fig.
2B, lanes 5, 8, and 14), the mutant with the
consensus site in the I-loop (designated
1I-loopH126N)
was not glycosylated (Fig. 2B, lane 11). Endoglycosidase H
(endoH) digestion removed the carbohydrate side chains and shifted
their electrophoretic mobility back to the mobility of the wt protein
(Fig. 2B, lanes 6, 9, and 15). Protease
protection assays have shown previously that the COOH-terminal domain
of the Cxs is located correctly outside the microsomes (7). Taken
together, these results demonstrated that the TM topology of Cx
polypeptides integrated into ER membranes (microsomes) was identical to
their topology in the PM, and therefore, a hypothetical aberrant TM
topology could not be responsible for the cleavage.
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Polypeptide Translocation Was Required for Cx Cleavage-- To determine whether Cx polypeptides have to be translocated into the lumen of the microsomes to be cleaved, we translated Cxs in the presence of microsomes that were preincubated with 0.25 and 1 mM NEM. This alkylating agent was previously reported to prevent protein translocation (23). To verify inhibition of protein translocation by NEM, Syn and PL were translated in parallel as control proteins. 0.25 mM NEM allowed the translocation of a portion of the control proteins, as indicated by a reduction in glycosylation of Syn, and a reduction of cleavage of pre-PL into PL. 1 mM NEM, however, completely abolished glycosylation of Syn, as well as cleavage of pre-PL, respectively (Fig. 3, lanes 1-3, middle and bottom panels). Successful targeting and binding of the newly translated polypeptides to the membrane was indicated by their recovery with the membranes after pelleting the microsomes through a 0.5 M sucrose cushion. Cx cleavage was completely abolished under the conditions described above (Fig. 3, lanes 1-3, top panel), indicating that membrane targeting was not sufficient for the Cx processing, but requires the translocation of the nascent Cx polypeptides into the membrane bilayer and into the lumen of the microsomes. Translocation and cleavage was also abolished when microsomes were preincubated at 30 °C for 30-90 min. This treatment resulted in the complete inactivation of the microsomal translocation activity (Fig. 3, lanes 5-7).
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Cxs Were Aberrantly Cleaved by SPase upon Integration into Microsomes-- A direct inhibitor for eucaryotic signal peptidases is not known, however, indirect experimentation reported in Falk et al. (7) suggested a cleavage by SPase. To obtain additional evidence for a SPase-mediated cleavage, microsomes were depleted of their soluble lumenal proteins by preincubating them at high pH (pH 9.5 and 10.2), using the protocol of Nicchitta and Blobel (19), followed by centrifugation through a 0.5 M sucrose cushion. However, protein depletion had little effect on the cleavage reaction (Fig. 3, lanes 8-10), indicating that the Cxs were not cleaved by a soluble, ER lumenal protease. Note that non-membrane integrated full-length Cxs generated as a by-product in the presence of microsomes (described above) were not removed prior to analysis, and therefore, are present on the gels shown in Fig. 3, lanes 8-12, and Figs. 5-7.
To exclude the possibility that the Cxs were cleaved by a different membrane-anchored ER protease, Cxs were translocated into microsomes that were preloaded at high pH with different class-specific protease inhibitors to ensure access of the chemicals to the microsomal lumen. While alkylating agents such as iodoacetamide (1 mM final concentration), or chelating agents such as EDTA (1 mM final concentration) efficiently blocked protein translation under the conditions described, preincubation of the microsomes in diisopropyl fluorophosphate (2 mM final concentration) or EGTA (2 mM final concentration), allowed translation, but had no significant influence on the cleavage reaction (Fig. 3, lanes 11 and 12). Furthermore, the cleavage was relatively independent of pH (~pH 5.5-9.0), temperature (30-41 °C), and RNA concentration (0.05-1 µg/10-µl reaction volume) (not shown). All these results provided additional strong evidence for a cleavage performed by SPase, and argued against a cleavage by any other known protease involved in protein degradation, such as "rapid ER degradation" (24) or "quality control" (25, 26).Cxs Were Cleaved at SPase "Cleavage Site Motifs" in Their First
Extracellular Domain--
The exact location of the cleavage sites was
mapped for cell-free expressed 1 and
1 Cx
polypeptides by NH2-terminal amino acid radiosequencing
after translating the Cxs in the presence of labeled phenylalanine
(Fig. 4A). Phenylalanine was
chosen because of its unique distribution within the
NH2-terminal portion of the Cx polypeptide. While no Phe
residues are present in the NH2-terminal domain, two Phe
residues separated by an isoleucine (Ile) are present in the first TM
domain (Phe-29 and Phe-31 in
1 Cx, and Phe-30 and Phe-32
in
1 Cx). Finally, single or double Phe residues are
present in the first extracellular loop (E1) (Phe-51 and Phe-68/Phe-69 in
1 Cx, and Phe-52 and Phe-70 in
1 Cx;
Fig. 4B). Radioactive peak activities released in the
individual degradation cycles (cycle 11 in the
1 Cx and
cycle 6 in the
1 Cx sequencing reaction) best matched
the distribution of Phe residues in the Cx sequences when aligned with
Phe-51 of
1 Cx and Phe-52 of
1 Cx,
respectively (Fig. 4, A and B). This analysis
identified two cleavage sites separated by five amino acid residues
from each other. Both sites were located within the border region
between TM1 and E1, between alanine (Ala) 40 and glutamic acid (Glu) 41 in
1 Cx, and glycine (Gly) 46 and aspartic acid (Asp) 47 in
1 Cx, respectively (Fig. 4A). Both
cleavage sites are homologous to natural SPase cleavage site motifs,
small, uncharged amino acid residues in positions
3 and
1 with
respect to the cleavage site (27), which are conserved in all Cx
isotypes at these positions (see Fig. 4B).
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The TM1 Domain and First Extracellular Loop (E1) Were Essential for
Cx Polypeptide Processing--
To determine which portion of the Cx
polypeptides contains the information that enables SPase to access the
cleavage sites, translation products of chimeric 1
Cx/Syn proteins (designated pCSR2 to pCSR5 in Fig.
6) (16) were analyzed after their
translation in the presence of microsomes. Syn is another
tetra-spanning membrane protein with an
Ncyt-Ccyt orientation with many similarities to the Cxs (22). In the chimeras, the Cx sequence (shown in
gray in Fig. 6) was progressively replaced from the COOH
terminus by Syn sequence (shown in black in Fig. 6).
Replacing COOH-terminal portions of the Cx sequence did not prevent the
cleavage (pCSR2 and pCSR3; Fig. 6, lanes 4 and
6), similar to the wt
1 Cx protein (Fig. 6,
lane 2). However, further replacing the second TM domain (TM2) and the first extracellular domain (E1) by Syn sequence abolished
the cleavage completely (pCSR4; Fig. 6, lane 8). Also, no
cleavage was observed for the chimera pCSR5 that consisted entirely of
Syn sequence except its NH2-terminal domain (Fig. 6,
lane 10), and in the wt Syn protein (Fig. 6, lane
12). N-Glycosylation of the Syn authentic glycosylation
sequence located within its first lumenal loop (22) occurred in wt Syn
(Fig. 6, lane 12), and in the chimera pCSR5 (Fig. 5,
lane 10) that both contain the first TM domain of Syn.
However, glycosylation did not occur in the chimera pCSR4 (Fig. 5,
lane 8) that also encodes the Syn authentic glycosylation
sequence, but instead contains the first TM domain of the
1 Cx. Taken together the results indicated that both, the TM1 and E1 domain, play an essential role for the access of SPase
to the cleavage sites in the wt Cxs.
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Increasing the Hydrophobic Character of the First TM Domain (TM1)
of Cxs Prevented Their Inappropriate Cleavage by SPase--
The
hydrophobic core region (h-region) of the TM1 domain of Cxs has a
length of only 18 amino acid residues, and in addition, it is
interrupted by an arginine (Arg) residue in its center that further
reduces the hydrophobic character of this region (see Fig.
4B). To determine if the weak hydrophobic character of this domain might be responsible for the Cx cleavage, three 1
Cx mutants were constructed in which the hydrophobicity of the TM1
domain was increased. In the mutant
1R32L, the central
Arg-32 residue was substituted with leucine (Leu). In the mutant
1+3L, three additional Leu residues were introduced
immediately adjacent to the central Arg-32 residue. In the mutant
1R32L+3L, the central Arg-32 residue was replaced by
leucine, and three additional Leu residues were introduced. Cleavage of
the mutants
1R32L, and
1+3L was
significantly reduced (Fig. 7,
lanes 4 and 6) compared with the wt
1 protein (Fig. 7, lane 2), and cleavage of
the mutant
1R32L+3L was completely abolished (Fig. 7,
lane 8). Membrane insertion and correct transmembrane
topology of the mutants was verified by their association with the
microsomal membranes after pelleting the microsomes through sucrose
cushions, and by their N-glycosylation after introducing
N-glycosylation consensus sites into the extracellular loops
E1 and E2 of the
1R32L+3L mutant (not shown).
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DISCUSSION |
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In this study we report that the synthesis of polytopic Cx membrane proteins in standard cell-free translation/translocation systems resulted in an aberrant proteolytic cleavage within their amino acid sequence that affected the entire pool of newly membrane integrated Cxs. These truncated Cxs differed from functional Cxs synthesized in tissues. This difference in Cx membrane integration in vitro and in vivo demonstrates that the membrane integration process of Cxs somehow differs from the membrane integration of other TM proteins.
The inability of the lysate system to generate normal, full-length
membrane integrated Cx polypeptides was unexpected since this system
has been found previously to accurately reproduce the biosynthesis of
many other membrane proteins including several examples of structurally
and functionally related polytopic membrane proteins, such as ion
channel subunits (28-31), aquaporins (32-34), and membrane pumps (35,
36). Subcellular Cx distribution studies (7, 37, 38) have indicated
that Cxs follow the secretory pathway from the ER, to the Golgi and PM,
suggesting that the ER-derived microsomal membranes used in the
cell-free expression system as target membranes represent the
appropriate intracellular membranes for Cx translocation. Also, the
cleavage cannot be explained by arguing that the microsomes were
derived from an inadequate source, since pancreatic acinar cells, from
which the microsomes were prepared (18), synthesize endogenous Cx
polypeptides (39, 40) and are able to integrate these proteins without
aberrant cleavage into their ER membranes (7). Finally,
N-glycosylation site tagging of 1 and
1 Cx (this study) combined with protease protection
assays (7) demonstrated that the TM topology of Cxs integrated in
vitro into microsomes corresponded to their natural TM topology
with NH2 and COOH termini correctly located outside the
microsomal vesicles (in the cytoplasm) and the two extracellular loops
located correctly inside the lumen of the microsomes. Therefore, an
inappropriate membrane topology can also be excluded as a possibility
that would lead to the Cx cleavage.
Amino acid sequencing of the cleaved Cxs identified cleavage sites at
the COOH-terminal end of the TM1 domain, located in the border region
between TM1 and the first extracellular loop (E1). Therefore, the
NH2-terminal domain and the TM1 domain was removed from the
rest of the Cx polypeptide sequence by this cleavage. Analysis of the
amino acid sequence adjacent to the cleavage sites showed that the
sites had homology to known cleavage site motifs (small, uncharged
amino acid residues in position 3 and
1 to the cleavage site) of
SPase (12, 27). They were identified at the COOH-terminal ends of
NH2-terminal ER targeting SP sequences of secretory and
transmembrane proteins (27) which are cleaved behind these motifs by
this protease. Several such
3/
1 motifs are conserved in the Cx
polypeptide isotypes in the border region between the TM1 and the E1
domains (compare Fig. 4B).
Analysis of the cleavage reaction further indicated that the cleavage was indeed performed by SPase. SPase is known to be present and active in microsomes (41). In Falk et al. (7) we reported that the cleavage occurred concomitant with the Cx polypeptide membrane integration, was strictly dependent on active, signal recognition particle-mediated protein translocation, and was restricted to the microsomal lumen. Here we report that the translocation of Cx/synaptophysin chimeras indicated that the TM1 domain and/or the first extracellular loop (E1) of the Cx sequence contained the cleavage signal and were essential for the cleavage reaction. Pretreatment of the microsomes with NEM, an alkylating agent that has been shown previously to prevent nascent chain translocation (23) indicated that insertion of the Cx polypeptides into the membrane was required for cleavage. Targeting and binding to the membrane bilayer was not sufficient. Depletion of the microsomes of their lumenal proteins had little effect on the cleavage reaction, suggesting that the cleavage was performed by a membrane-bound ER protease. Finally, several class-specific protease inhibitors had no effect on the cleavage reaction, even under conditions where direct access of the inhibitors to the lumen of the microsomes was ensured. SPase, in contrast to other known ER lumenal proteases is not affected by known protease inhibitors, a property attributed to its unusual cleavage mechanism (42). All these properties are indicative of a cleavage by SPase falsely performed on the Cx polypeptide sequence by this enzyme in vitro. Inappropriate uncovering and processing of "cryptic" SPase cleavage sites has been reported previously for some membrane proteins after modifying the amino acid sequence in or near the hydrophobic core region of their signal sequences (43-46). However, no inappropriate processing of a wt protein, as in the case of the Cxs, has been reported. Why then is the lysate system not able to generate full-length membrane integrated Cx polypeptides, and SPase efficiently recognizes and cleaves the Cx polypeptides within their functional sequence when they are translocated in vitro?
In the Cxs, the TM1 domain functions as an internal SA
sequence,2 suggesting that
the cleavage could be due to an incorrect recognition of this domain as
a cleavable SP sequence. Sequence comparison indicated a principal
structural similarity between cleavable SP sequences and internal SA
sequences. Both sequences consist of a short, positively charged
NH2-terminal region (n-region), a central hydrophobic core
region (h-region), and a more polar COOH-terminal region (c-region)
(47, 48); and many SA sequences of membrane proteins are followed by
one or more 3/
1 cleavage site motifs, although they are not
processed in detectable amounts by SPase in vitro or
in vivo. However, an obvious difference between SP and SA
sequences is the considerably longer h-region of the latter. While
h-regions of cleaved SP sequences are typically 7-15 residues long and
are rarely longer than 17 residues, the h-regions of uncleaved SA
sequences range from 20 to 30 residues in length (48, 49). Nilsson
et al. (48) found that the length of the h-region is
directly decisive for its recognition as a SP or SA sequence. The
different length results in a different positioning of SP and SA
sequences within the translocon during the translocation process,
allowing SPase to access and cleave NH2-terminal SP
sequences, while the cleavage of internal SA sequences is prevented. A
sequence of 17-19 hydrophobic residues was determined as the critical
length (48). In Cxs the hydrophobic core of the TM1 domain has a length
of only 18 residues. In addition, it contains an arginine residue in
its center that interrupts the hydrophobic character of this domain
(see Fig. 4B).
To test the hypothesis that the relatively weak hydrophobic character
of the Cx TM1 domain may cause its false recognition and positioning,
1 Cx mutants with an increased hydrophobic character of
their TM1 domain were generated. Replacing the central arginine residue
with leucine, or elongating the h-region by 3 leucine residues to 21 residues already drastically reduced the cleavage efficiency.
Increasing the hydrophobicity further by replacing the central
arginine, and in addition, elongating the h-region by 3 leucine
residues into a homogenous stretch of 21 hydrophobic residues
completely abolished the cleavage (Fig. 7). These results indicate that
the SA sequence of wt Cxs is inappropriately recognized and positioned
as a cleavable SP sequence when translated in vitro, due to
its relatively weak hydrophobic character. The inappropriate cleavage
can be overcome by increasing the hydrophobic character and the length
of this domain (represented schematically in Fig. 8, A and B).
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These findings, however, do not explain why the SA sequence of Cxs was not appropriately recognized in vitro. A mechanism that would explain this phenomenon is that a specific factor, such as a chaperone that normally is involved in the membrane integration of Cxs and, for example, binds to the Cx polypeptide sequence to aid in their correct positioning within the ER translocon (represented schematically in Fig. 8C), is absent or not functional in the cell-free expression system. This hypothesis is supported by our findings obtained with the expression of Cxs in transfected tissue culture cells (7). Overexpression of Cxs in different cell types resulted in the cleavage of a portion of the newly synthesized Cx polypeptides upon their translocation into the ER membranes that correlated with the expression level. This result suggests that a factor involved in their proper membrane integration has only a limited availability within the cell. Further experimentation will be aimed at the determination of the components that provide an appropriate positioning of the Cx polypeptides in the translocon to facilitate the synthesis of full-length membrane integrated Cxs in vivo.
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ACKNOWLEDGEMENTS |
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We thank Nalin Kumar, Richard Scheller, Dieter Zopf, Peter Walter, and Rudolph Leube for generously providing cDNA clones and connexin chimeras, Lisa Bibbs and The Scripps Research Institute's Core Facility for protein sequencing, Angelika Kehlenbach and Jutta Falk-Marzillier for critically reading the manuscript, Jialing Lin and members of the Gilula laboratory for helpful discussions.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant GM 37904, a grant from the Lucille P. Markey Charitable Trust (to N. B. G.), and a Deutsche Forschungsgemeinschaft fellowship (to M. M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Dept. of Cell
Biology, MB6, The Scripps Research Institute, 10550 North Torrey Pines
Rd., La Jolla, CA 92037. Tel.: 1-619-784-2352; Fax: 1-619-784-2345; E-mail: MFalk{at}scripps.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; Cx, connexin; PM, plasma membrane; SA, signal anchor; PAGE, polyacrylamide gel electrophoresis; SP, signal peptide; SPase, signal peptidase; TM, transmembrane; wt, wild type; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; NEM, N-ethylmaleimide; PL, prolactin.
2 M. Falk, unpublished data.
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