(Received for publication, February 22, 1995; and in revised form, June 19, 1995)
From the
Secretory proteins are synthesized with a signal sequence that is usually cleaved from the nascent protein during the translocation of the polypeptide chain into the lumen of the endoplasmic reticulum. To determine the fate of a cleaved signal sequence, we used a synchronized in vitro translocation system. We found that the cleaved signal peptide of preprolactin is further processed close to its COOH terminus. The resulting fragment accumulated in the microsomal fraction and with time was released into the cytosol. Signal sequence cleavage and processing could be reproduced with reconstituted vesicles containing Sec61, signal recognition particle receptor, and signal peptidase complex.
Signal sequences mediate the entry of proteins into the
secretory pathway (Blobel and Dobberstein, 1975a). As soon as they
emerge from the ribosome, signal sequences are recognized by the
cytosolic signal recognition particle (SRP) ()(Walter et
al., 1981), which targets the nascent proteins to the membrane of
the endoplasmic reticulum (ER). Specific binding of the
ribosome-nascent chain-SRP complex to the ER membrane occurs through
binding to the membrane-bound SRP receptor (also called docking
protein) (Gilmore et al., 1982; Meyer et al., 1982).
After SRP displacement and insertion of the nascent chain into the
translocation complex, the signal sequence is cotranslationally cleaved
(Blobel and Dobberstein, 1975b), and the mature part of the protein is
translocated into the lumen of the ER.
Although signal sequences display almost no sequence similarities, they share some common features. They can be found at the amino terminus of the respective proteins and consist in most cases of 20-30 amino acid residues. Furthermore, they usually show a characteristic tripartite structure; a positively charged amino-terminal region precedes a central hydrophobic core, which is followed by a COOH-terminal polar region that contains the cleavage site for the signal peptidase (von Heijne, 1985).
The microsomal signal peptidase has been purified as a complex of five subunits (signal peptidase complex (SPC)) with apparent molecular masses of 12, 18, 21, 22/23, and 25 kDa (Evans et al., 1986). The 18-kDa and the 21-kDa subunits, SPC18 and SPC21, are mammalian homologues of the Escherichia coli leader peptidase (van Dijl et al., 1992), which performs the signal sequence cleavage as a single protein (Zwizinski and Wickner, 1980). Both subunits are also homologous to the yeast SEC11 protein (Greenburg et al., 1989; Shelness and Blobel, 1990), which is an essential component of the signal peptidase complex in Saccharomyces cerevisiae (Böhni et al., 1988). All leader peptidase homologues contain a highly conserved serine residue that is supposed to catalyze the actual signal peptidase reaction (Sung and Dalbey, 1992).
In E. coli, cleaved signal peptides are digested by signal peptide peptidases (Hussain et al., 1982). This degradation is thought to be initiated by membrane-bound protease IV, which cleaves the peptide within the hydrophobic core (Novak and Dev, 1988). The resulting fragments can be released into the cytosol and further hydrolyzed by oligopeptidase A (Novak and Dev, 1988). Both signal peptide peptidases are endoproteases and account for the majority of signal peptide degrading activity in vitro (Novak et al., 1986).
Most likely, signal peptides are also degraded in eukaryotic cells (Habener et al., 1979), but the process itself, the enzymes involved, and the sites of cleavage have not yet been elucidated. We show here that the preprolactin (PPL) signal peptide is further processed in rough microsomes. The resulting COOH-terminal signal peptide fragment could be detected in the cytosolic fraction of translocation assays. Further results suggest that signal peptide processing is required although not sufficient for the release of the signal peptide from the membrane.
After translation of PPL86, samples were put on ice, and the
salt concentration was raised to 500 mM KOAc. The samples were
incubated for 5 min on ice and layered on top of a 50-µl cushion
containing 500 mM sucrose, 500 mM KOAc, 50 mM Hepes-KOH, pH 7.9, 5 mM Mg(OAc), and 1 mM dithiothreitol. Membranes were pelleted by a 3-min centrifugation
at 48,000 rpm and 4 °C in a Beckman TLA 100 rotor. Reconstituted
vesicles were sedimented twice through a cushion containing only 100
mM sucrose under otherwise identical conditions. The
supernatants were removed, and the pellets were resuspended in a buffer
containing 20 mM Hepes-KOH, pH 7.9, 70 mM KOAc, 10
mM KCl, 3 mM Mg(OAc)
, 200 µM GTP, and 1 mM dithiothreitol. Nascent chains were
released from ribosomes by the addition of puromycin, pH 7.9, to the
final concentration of 1 mM and by a 12-min incubation at 28
°C. Membranes were pelleted by centrifuging translocation assays
for 3 min at 48,000 rpm and 4 °C in a Beckman TLA 100 rotor.
Proteinase K treatment was performed by incubating translocation assays for 10 min at 25 °C in the presence of 300 µg/ml proteinase K. For controls, proteinase K was omitted or added in the presence of 1% Triton X-100.
PPL signal peptide-specific molecular weight markers were produced by in vitro transcription/translation of PPL TAG18, PPL TAG25 (High et al., 1993), and PPL30 (synthesized by polymerase chain reaction amplification according to Nilsson et al. (1994)).
Figure 1:
Characterization of PPLMM. A, primary structure of the signal sequences of PPL and PPLMM.
Altered amino acids are shown in boldface. B, signal
sequence cleavage of PPL and PPLMM. PPL wild type mRNA (lanes1 and 2) or PPLMM mRNA (lanes3 and 4) was translated in the absence (lanes1 and 4) or presence (lanes2 and 3) of rough microsomes (RM).
[S]Methionine-labeled translation products were
separated by SDS-polyacrylamide gel electrophoresis and visualized by
phosphoimaging.
Figure 2: Puromycin release of ribosome-bound PPL86. A, PPLMM86 mRNA (lanes1-4) or PPL86 mRNA (lane5) was translated in the presence of SRP and RM. After translation, membranes were sedimented through a high salt sucrose cushion. The supernatant (SN, lane1) was removed, and the pellet (P, lane2) was resuspended and incubated with 4 mM cycloheximide (CHI, lane3) or 1 mM puromycin (Puro, lane4). B, quantification of the results shown in panel A, lanes3-5. The amount of radioactivity in the area of PPL85, PL56, and PSP was determined by using the phosphoimager as described under ``Experimental Procedures.'' The amount of radioactivity in unprocessed PPL86 (A, lane2) was taken as 100%.
When nascent chains are released from the ribosomes by puromycin, they become translocated across the microsomal membrane (Redman and Sabatini, 1966), and their signal sequence is cleaved. Accordingly, we detected two peptides with apparent molecular masses of about 5 and 3 kDa (Fig. 2A, lanes4 and 5) representing the mature part of PPL86 (PL56) generated by signal sequence cleavage between amino acid residues 30 and 31 (Sasavage et al., 1982) and the processed signal peptide (PSP), respectively. Processing was only observed when puromycin was added and did not occur in the presence of cycloheximide (Fig. 2A, lane3). The ratios of label in the 5- and 3-kDa peptides were roughly 1:1 for the MM mutant (Fig. 2B, lane4) and 3:1 for the wild type (Fig. 2B, lane5). These ratios are consistent with the methionine content of PL56 (three methionines) and of the processed signal peptide (three methionines in MM mutant, one in wild type).
Figure 3: Localization of the processed signal peptide. A, proteinase K treatment of microsomal membranes. Microsomes containing puromycin-released PPLMM86 (lane1) were incubated with proteinase K in the absence (lane2) or presence (lane3) of Triton X-100. B, pelleting of microsomes. Assays containing rough microsomes and membrane-inserted PPLMM86 were centrifuged directly (lanes1-3) or after treatment with puromycin (lanes4-6) and separated into supernatant (lanes2 and 5) and pellet (lanes3 and 6).
When membranes were pelleted after puromycin release, the PSP was found in the supernatant (Fig. 3B, lane5), whereas PL56 was found in the microsomal pellet (Fig. 3B, lane6). We therefore conclude that the PSP is released into the cytosol.
Figure 4:
Characterization of the processed signal
peptide. A, parallel electrophoretic analysis of
puromycin-released wild type PPL86 (lane2) and
marker peptides (lanes1 and 3). B,
cysteine labeling of PPL86 and its processed products. PPLMM86 mRNA was
translated in the presence of [S]cysteine (lanes1 and 2) or
[
S]methionine (lane3). The
assay was performed as described for Fig. 2A.
This conclusion was confirmed by labeling PPL86 with
[S]cysteine. The signal sequence of PPL contains
one cysteine at position 25, and PL56 contains two cysteines. If the
signal peptide were truncated amino-terminally of cysteine 25, the PSP
should not be detectable on gels. Indeed, after puromycin treatment of
cysteine-labeled PPL86, PL56 accumulated, whereas the PSP remained
invisible (Fig. 4B, lane2).
Figure 5: Pulse-chase experiment. Nascent PPLM86 was released from the ribosomes by puromycin and incubated at 25 °C. Aliquots were taken at the time points indicated and separated into supernatant (SN) and pellet (P). Lane15 shows in vitro synthesized intact signal peptide (PPL30).
Figure 6: Membrane translocation and signal sequence cleavage by reconstituted vesicles. A, protein composition of the SPC preparation used for reconstitution. The proteins were separated on a 16% acrylamide gel and stained with Coomassie Brilliant Blue. B, translocation into reconstituted vesicles. PPL mRNA was translated in the presence of SRP and membrane buffer (lanes1 and 4) or reconstituted vesicles containing Sec61 and SRP receptor (lanes2 and 5) or reconstituted vesicles containing Sec61, SRP receptor, and SPC (lanes3 and 6). After translation, half of the samples were treated with proteinase K (lanes4-6).
To assay for signal peptide processing by the reconstituted vesicles, we used membrane-inserted and puromycin-released PPL86. When reconstituted vesicles containing only Sec61 and SRP receptor were used, no processing of PPLMM86 was observed (Fig. 7A, lane1). When reconstituted vesicles containing Sec61, SRP receptor, and SPC were used, a peptide of about 5 kDa and, similar to early steps in microsomes (Fig. 5, lane4), two peptides of about 3 kDa were generated (Fig. 7A, lane2). The 5-kDa peptide and the faster migrating 3-kDa peptide comigrated with PL56 and the PSP, respectively (Fig. 7B, lanes4 and 5). However, the SP* is not the intact signal peptide, as it exhibits a slightly higher electrophoretic mobility when compared to in vitro synthesized intact signal peptide (PPL30, Fig. 7C). Thus, reconstituted vesicles can process the signal sequence; however, the processing sites differ from those in microsomes. Furthermore, the considerable amount of SP* suggests that signal peptide processing is inefficient in reconstituted vesicles.
Figure 7: Processing of PPL86 by reconstituted vesicles. A, puromycin release of PPLMM86 bound to reconstituted vesicles with (lane2) or without (lane1) SPC. B, PPLMM86 mRNA was translated in the presence of SRP and reconstituted vesicles containing Sec61, SRP receptor, and SPC (lanes1-4) or rough microsomes (lane5). After translation, membranes were sedimented through a high salt sucrose cushion. The supernatant (lane1) was removed, and the pellet (lane2) was resuspended and incubated with 4 mM cycloheximide (lane3) or 1 mM puromycin (lane4). C, comparison of puromycin-released PPLMM86 from reconstituted vesicles (lane2) with marker peptides (lanes1 and 3).
When reconstituted vesicles were pelleted after puromycin treatment, the PSP was found in the supernatant (Fig. 8, lane5), whereas PL56 and the SP* were detected in the pellet (Fig. 8, lane6). This suggests that the PSP is released into the cytosol by reconstituted vesicles, whereas the SP* is associated with the membranes.
Figure 8: Localization of processed signal peptides in reconstituted vesicles. Assays containing reconstituted vesicles and membrane-inserted PPLMM86 were centrifuged directly (lanes1-3) or after treatment with puromycin (lane4-6) and separated into supernatant (lanes2 and 5) and pellet (lanes3 and 6).
The characterization of the fate of signal sequences after
their cleavage from nascent polypeptides is difficult for several
reasons: 1) no antibodies could yet be raised against any signal
peptide, and thus their identification presents a major problem; 2)
signal peptides are small and have to be distinguished from small
peptides accumulating in translation systems as a result of premature
chain termination; 3) signal peptides can usually not be labeled
efficiently as they contain only few methionine or cysteine residues
for labeling with S; and 4) signal peptides are probably
very rapidly further processed, making it necessary to identify also
fragments derived from processing reactions.
The PPL signal peptide fragment generated by microsomes and released into the cytosol comprises the amino-terminal, roughly 20 amino acid residues of the signal sequence. The approximate size was estimated from a comparison with defined amino-terminal PPL signal peptide fragments as standards. According to our size estimation, cleavage occurs between the two leucine clusters in the middle of the hydrophobic core of the PPL signal peptide (see Fig. 1A). This indicates that the mammalian signal peptide peptidase like its E. coli counterpart cleaves the signal peptide in the hydrophobic core.
After short incubation with puromycin, the apparently intact SP accumulated and was processed to the PSP over time. Initially, the SP as well as the PSP were associated with the microsomes. Only after prolonged incubation, the PSP was released into the supernatant, whereas the SP stayed in the pellet. This suggests that cleavage within the hydrophobic core of the signal peptide is required although not sufficient for its release from the membrane and indicates that the PSP undergoes a time-dependent release into the cytosol. The release of the PSP might be important for subsequent polypeptide translocation across the membrane, a notion being supported by the finding that inhibition of E. coli signal peptide peptidase results in inhibition of translocation (Chen and Tai, 1989). Degradation of signal peptides might thus contribute to the maintenance of fast and efficient protein translocation across the ER membrane.
It has been shown before that the addition of prepromelittin to vesicles reconstituted from rat liver microsomes resulted in the generation of the intact signal peptide (Mollay et al., 1982). This finding does not contradict our results, as prepromelittin signal sequence cleavage occurred from polypeptides not inserted into the membrane, and signal peptides accumulating in the cytosol could have escaped further processing by signal peptide peptidase. Furthermore, an extraction procedure was used that specifically selects for hydrophobic peptides (Mollay et al., 1982), thus excluding processed signal peptide fragments from detection.
Reconstituted vesicles containing SPC were able to process the
signal peptide to the PSP, which was released into the supernatant.
This suggests that the reconstituted vesicles containing SPC contained
also signal peptide peptidase activity. Signal peptide processing was,
however, found to be less efficient with reconstituted vesicles than
with microsomes. Whether the signal peptide peptidase is one of the
known subunits of the SPC or partially copurified with one of the
components used for reconstitution remains to be determined. Signal
peptide peptidase from E. coli has been characterized before
(Ichihara et al., 1986). It shows no homologies to any one of
the SPC subunits (Shelness et al., 1988; Greenburg et
al., 1989; Shelness and Blobel, 1990; Greenburg and Blobel, 1994).
This includes SPC12, of which the cDNA has been recently cloned and
sequenced. ()
Figure 9: Compilation of so far characterized signal peptide fragments. The localization of the fragments is indicated below the description of the source signal peptide. A, signal peptide fragments found associated with MHC class I molecules. Data are from Wei and Cresswell(1992) and Henderson et al.(1992). B, signal peptide fragment characterized in this study (dots indicate that the COOH-terminal end of the fragment is estimated from comparison with marker peptides) (see Fig. 4A).
Figure 10: Schematic illustration of signal sequence cleavage and processing in rough microsomes. After membrane insertion of the nascent chain (1), the signal sequence is cleaved by signal peptidase (2), and the signal peptide is further processed by signal peptide peptidase (3). The signal peptide fragments are then released from the translocation complex either to the cytosol or into the ER lumen (4) to allow a new round of translocation. Proteins at the translocation site are shaded, and enzyme activity is indicated by asterisks.