(Received for publication, September 22, 1995; and in revised form, November 28, 1995)
From the
The cleavage of signal sequences of secretory and membrane proteins by the signal peptidase complex occurs in the lumen of the endoplasmic reticulum. Mammalian signal peptidase consists of five subunits. Four have been cloned, SPC18, SPC21, SPC22/23, and SPC25, of which all but SPC25 have been demonstrated to be single-spanning membrane proteins exposed to the lumen of the endoplasmic reticulum.
We have determined the cDNA sequence of the remaining 12-kDa subunit (SPC12) as well as the membrane topologies of SPC12 and SPC25 in rough microsomes. Both polypeptides span the membrane twice with their N and C termini facing the cytosol and contain only very small, if any, lumenal domains. Therefore, SPC12 and SPC25 are likely to be involved in processes other than the enzymatic cleavage of the signal sequence.
Translocation of polypeptide chains across the endoplasmic
reticulum (ER) ()membrane is triggered by signal sequences
(Blobel and Dobberstein, 1975a). In the cotranslational mode of
translocation the signal sequence is initially recognized in the
cytosol by the signal recognition particle (Walter et al.,
1981). Subsequently, signal recognition particle interacts with its
membrane receptor (Gilmore et al., 1982; Meyer et
al., 1982), and the ribosome-bound nascent chain is targeted to
the ER where it is transferred into a protein-conducting channel (for
review, see Walter and Johnson(1994)). At some point, a second signal
sequence recognition event takes place in the membrane (Jungnickel and
Rapoport, 1995). How the signal sequence is recognized during the
posttranslational mode of translocation is presently unknown. However,
it has been speculated that Sec72p (Feldheim and Schekman, 1994) or
Sec62p (Johnsson and Varshavsky, 1994) are somehow involved in this
process.
During the next step, translocation of the nascent chain through the membrane, the signal sequence of most secretory and membrane proteins is cleaved off. Cleavage occurs by the signal peptidase complex (SPC) as soon as the lumenal domain of the translocating polypeptide is large enough to expose its cleavage site to the enzyme (Blobel and Dobberstein, 1975b). The signal peptidase complex is possibly also involved in proteolytic events in the ER membrane other than the processing of the signal sequence, for example the further digestion of the cleaved signal peptide (Lyko et al., 1995) or the degradation of membrane proteins (Mullins et al., 1995).
Mammalian signal peptidase has been purified from dog pancreas microsomes as a complex of five different polypeptide chains (Evans et al., 1986). The cDNAs of four of the subunits (SPC18, SPC21, SPC22/23, SPC25) have been cloned and sequenced (Shelness et al., 1988; Greenburg et al., 1989; Greenburg and Blobel, 1994). SPC18 and SPC21 are single-spanning membrane proteins; the majority of both proteins is located within the lumen of the ER and contains a second only moderately hydrophobic sequence (Shelness et al., 1993). Both subunits show high homology to each other (Shelness et al., 1988). Moreover, they are related in sequence to leader peptidase (van Dijl et al., 1992), the enzyme responsible for signal sequence cleavage during translocation of proteins across the plasma membrane of bacteria (Zwizinski and Wickner, 1980). These polypeptides may therefore function as catalytic subunits. SPC22/23 has an identical topology to SPC18 and SPC21. It is also a single-spanning membrane protein with a second only somewhat hydrophobic segment located in the lumen of the ER (Shelness et al., 1993). SPC22/23 contains an N-glycosylation site, and its migration in SDS-gels as two bands of 22 and 23 kDa, respectively, is likely to arise from differential trimming of its oligosaccharide moiety (Evans et al., 1986). SPC25 has recently been cloned. It again contains two hydrophobic sequences and was proposed to be a single-spanning membrane protein as well, with a large N-terminal domain in the lumen of the ER (Greenburg and Blobel, 1994). However, its membrane topology has not yet been directly determined.
In this paper, we present the sequence of the remaining subunit of the mammalian signal peptidase complex (SPC12) as well as the membrane topologies of SPC12 and SPC25. Surprisingly, both polypeptides are largely exposed to the cytosolic compartment and have very few, if any, amino acid residues in the lumen of the ER. Since this is where the active site of the enzyme must be located, SPC12 and SPC25 are not likely to be directly involved in signal sequence cleavage.
Figure 1: Sequence of human SPC12. The human nucleotide and deduced amino acid sequences are shown. Peptides obtained from purified dog SPC12 are aligned above in one-letter codes; amino acid residues that differ from the human sequence are indicated in three-letter codes. Predicted membrane-spanning segments are double underlined. The peptide used to raise antibodies is indicated with asterisks above.
Figure 2:
Accessibility of SPC12 and SPC25 to
proteases. Ribosome-stripped microsomes were incubated at the indicated
temperatures for 15 min and then subsequently cooled down to 0 °C.
Proteolysis was carried out with 250 µg/ml proteinase K for 1 h on
ice. As indicated, 1% Triton X-100 was added before starting
proteolysis. The samples were precipitated with 20% trichloroacetic
acid and analyzed by SDS-PAGE and immunoblotting. The following
peptide-specific antibodies were used: TRAP, N terminus; SPC22/23,
C terminus; SPC25, C terminus; SPC12, N terminus. The stars indicate degradation products of
TRAP
.
If microsomes were treated in the cold with proteinase K,
SPC12 migrated in SDS gels with an apparent molecular mass of 11 kDa,
in contrast to 12 kDa for the undigested protein, and showed an
unchanged reactivity toward the antibody (Fig. 2, lane
2). The same results were observed when trypsin was used instead
of proteinase K (data not shown). The N-terminal antigenic peptide of
SPC12 does not contain a trypsin cleavage site. For all of these
reasons, it is clear that a portion of the C terminus was cut off, and
thus this region must be located in the cytosol, accessible to the
protease. Surprisingly, if the digestion was carried out after
solubilization of the microsomes in Triton X-100, no further
degradation was observed (Fig. 2, lane 3). A control
protein, TRAP (originally called SSR
), a single-spanning
membrane protein with only few C-terminal residues located in the
cytosol (Hartmann et al., 1993), did not change significantly
in size after proteinase K treatment but was completely digested if
Triton X-100 was also added (lanes 2 and 3). On the
other hand, SPC22/23 behaved similarly to SPC12 in that its
accessibility to proteases was the same before and after solubilization
of the membranes. In both cases, the mobility of SPC22/23 was unchanged
after protease digestion (lanes 2 and 3). The
remarkable protease resistance of SPC18, SPC21, and SPC22/23 has been
reported before (Shelness et al., 1993). Therefore, it seems
that the signal peptidase is a stable complex of polypeptide chains
remaining almost resistant to protease treatment even after
solubilization of the membranes.
We reasoned whether this unusual
behavior could be exploited to determine the localization of the N
terminus of SPC12, which is recognized by the antibody. We pretreated
microsomes at elevated temperatures with the hope that the tight
interaction between the signal peptidase subunits can be broken by
heat. The subsequent protease digestion was carried out on ice in the
absence or presence of Triton X-100. As shown in Fig. 2(lanes 6-9), pretreatment at 50 or 60
°C allowed the complete degradation of the N terminus of SPC12,
even in the absence of detergent. In contrast, TRAP and SPC22/23
were only digested in the presence of Triton X-100, demonstrating that
these proteins have maintained their membrane orientation with a major
lumenal domain and that the membrane integrity was not compromised by
the heat treatment. These data confirm the membrane topologies of
TRAP
and SPC22/23 and show that both the N and C termini of SPC12
are located in the cytoplasmic compartment. Such a model is in good
agreement with the predicted membrane topology of SPC12 having two
adjacent membrane-spanning segments.
Figure 3: Sequence of canine SPC25. The dog amino acid sequence is shown (Greenburg and Blobel, 1994). Proposed membrane anchors are double underlined. Peptides used to raise antibodies are indicated with asterisks above.
The mammalian signal peptidase complex consists of five subunits (Evans et al., 1986), and, with the data on SPC12 and SPC25 presented here, the sequences and membrane topologies of all subunits are now known (Fig. 4). The primary structure of SPC12 is not related to any of the other four signal peptidase subunits (SPC18, SPC21, SPC22/23, and SPC25) and shows no homology to any known protein in the data bases. SPC12 contains two hydrophobic segments close to each other. As in the case of SPC25, the hydrophobicity of either segment is substantially higher than that of the second somewhat hydrophobic regions found in SPC18, SPC21, and SPC22/23. This suggested to us that SPC12 and SPC25 must span the membrane twice. To test this, dog pancreatic microsomes were treated with proteinase K, and the degradation of both polypeptides was followed using affinity-purified peptide-specific antibodies. Indeed, SPC12 and SPC25 each contain two adjacent membrane anchors with both their N and C termini oriented toward the cytosolic compartment (Fig. 2). Surprisingly, both subunits expose only very few, if any, amino acids to the lumen of the ER where the catalytic site of the signal peptidase complex must be positioned. Our data clearly contradict the recent assumption that SPC25 is a single-spanning membrane protein with an extended N-terminal segment inside the ER lumen (Greenburg and Blobel, 1994). The reported disulfide bridge in the native protein (Greenburg and Blobel, 1994) could be a linkage between two of the three cysteins present in the membrane anchors as has been found in the leader peptidase of the Escherichia coli plasma membrane (Whitley et al., 1993).
Figure 4: Membrane topology of mammalian signal peptidase subunits. The topologies of all five signal peptidase subunits are schematically illustrated. The gray boxes indicate the membrane-spanning domains of the polypeptides. SPC 22/23 is drawn in the glycosylated form.
Interestingly, SPC12 and SPC25 remained almost protease
resistant even when the membranes were solubilized with detergent
before adding the protease. The same phenomenon has previously been
described for SPC18, SPC21, and SPC22/23 (Shelness et al.,
1993). Thus, the five polypeptides of the mammalian signal peptidase
seem to form a very tight complex whose core is resistant to protease
treatment even after solubilization of the membrane. Our results
further indicate that the compact structure of the signal peptidase
complex can be broken by heat treatment. After incubation of the
microsomes at 50 or 60 °C, additional regions of SPC12, SPC22/23,
and SPC25 became accessible to the added protease. Heat pretreatment of
the microsomes did not influence the membrane orientation of the two
control proteins, SPC22/23 and TRAP. These data are in good
agreement with results of other groups. Fujimoto et al.(1984)
had found that an incubation of partially purified SPC at 40 °C
slightly decreased its enzyme activity and that further increase of the
temperature inactivated the enzyme. Shelness et al.(1993)
observed that SPC18, SPC20, and SPC22/23 were more accessible to
proteases after treating a detergent extract of microsomes at 75
°C.
What might be the functions of the different signal sequence subunits? Because of their homology to the leader peptidase of bacteria, SPC18 and SPC21 may function as catalytic subunits and are believed to represent members of a novel protease family, which have a serine as a key amino acid in the active site (van Dijl et al., 1992; Dalbey and von Heijne, 1992; van Dijl et al., 1995). The membrane topologies of SPC18 and SPC21 fit with this hypothesis as the majority of both polypeptides, including the proposed catalytic center, are exposed to the lumen of the ER (Shelness et al., 1993). Even though SPC22/23 has the same membrane orientation, a direct contribution to signal sequence cleavage seems unlikely as it has no homology to known proteases. However, it is possible that this polypeptide represents a new protease with an unknown substrate specificity. Alternatively, it has been speculated that SPC22/23 may be involved in substrate binding (Shelness et al., 1988).
The membrane topology of SPC12 and SPC25 is quite unexpected as neither protein exposes a substantial domain to the lumen of the ER. Therefore, SPC12 and SPC25 are likely to be involved in processes other than substrate binding or the actual enzymatic reaction. What then could be the functions of SPC12 and SPC25? The extended cytosolic regions of SPC12 and SPC25 may interact with soluble factors or with domains of membrane proteins facing the cytosolic compartment. One could speculate that both polypeptides may be involved in fixing the signal peptidase in a distinct position to the translocation channel or in recruiting the entire enzyme complex to the translocation site when the translating ribosome becomes membrane bound. Moreover, it is also possible that the four membrane-spanning segments of SPC12 and SPC25 are involved in the transfer of the signal sequence from its insertion site to the active center of the signal peptidase.