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• W2139833905 abstract "Article15 November 1997free access Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin Bruno Martoglio Corresponding Author Bruno Martoglio Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany Search for more papers by this author Roland Graf Roland Graf Laboratorium für Biochemie II, ETH-Zentrum, Universitätstrasse 16, 8092 Zürich, Switzerland Search for more papers by this author Bernhard Dobberstein Bernhard Dobberstein Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany Search for more papers by this author Bruno Martoglio Corresponding Author Bruno Martoglio Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany Search for more papers by this author Roland Graf Roland Graf Laboratorium für Biochemie II, ETH-Zentrum, Universitätstrasse 16, 8092 Zürich, Switzerland Search for more papers by this author Bernhard Dobberstein Bernhard Dobberstein Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany Search for more papers by this author Author Information Bruno Martoglio 1, Roland Graf2 and Bernhard Dobberstein1 1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Postfach 106249, 69052 Heidelberg, Germany 2Laboratorium für Biochemie II, ETH-Zentrum, Universitätstrasse 16, 8092 Zürich, Switzerland The EMBO Journal (1997)16:6636-6645https://doi.org/10.1093/emboj/16.22.6636 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now. Introduction Secretory proteins and membrane proteins contain a signal sequence for targeting to the protein-conducting channel and subsequent translocation across or insertion into the endoplasmic reticulum (ER) membrane (Rapoport et al., 1996). During transport into the ER lumen the signal sequence is often cleaved from the precursor protein by the signal peptidase (Blobel and Dobberstein, 1975). The characteristic feature of a signal sequence is a tripartite structure: a polar N-terminal n-region, a hydrophobic core (h-region) of 7–15 residues and a polar C-terminal c-region that contains the consensus sequence for signal peptide cleavage (von Heijne, 1985). The n-region of most signal sequences comprises only a few residues. However, some signal sequences have extended n-regions, of up to 150 residues. The function of such long n-regions is not as yet known. The fate of only a few signal sequences has been elucidated. Fragments derived from the signal sequence of some secretory proteins or type I membrane proteins have been found associated with MHC class I molecules and are transported to the cell surface for presentation to cytolytic T cells. Some signal peptide fragments (SPFs) corresponding mainly to C-terminal segments of the respective signal sequence become associated with MHC class I molecules independent of the transporters associated with antigen processing (TAP) (Henderson et al., 1992; Wei and Cresswell, 1992). However, for one SPF derived from the n-region of the lymphocytic choriomeningitis virus envelope protein a strictly TAP-dependent binding to MHC class I molecules has been reported (Hombach et al., 1995). These results indicate that SPFs can be released from the membrane to the ER lumen or to the cytosol. Besides functioning in antigen presentation, nothing is known about the physiological roles of SPFs released into the cytosol or the ER lumen. Using a synchronized in vitro system we have previously shown that the cleaved signal peptide of the secretory protein hormone preprolactin (p-Prl) is further processed in the ER membrane and that the resulting N-terminal SPF is released into the cytosol (Lyko et al., 1995). Processing of the cleaved signal sequence was found to be sensitive to the immunosuppressive proline isomerase inhibitor cyclosporin A (Klappa et al., 1996). Cyclosporin A is known to bind to cellular proteins termed cyclophilins which have proline isomerase activity and are thought to modulate the activity of various enzymes (Schreiber and Crabtree, 1992). It is thus conceivable that a cyclophilin in the ER regulates signal sequence processing and subsequent release of the SPF into the cytosol. To determine possible functions of SPFs released into the cytosol, we followed the fate of the p-Prl signal sequence and identified components interacting with the cleaved signal sequences in the membrane and the SPF in the cytosol using site-specific photo-crosslinking (Martoglio and Dobberstein, 1996). We found that in the cytosol the p-Prl SPF interacts efficiently with calmodulin (CaM). The p-Prl signal sequence has an extended basic n-region such that it can potentially form a basic amphipathic α(baa)-helix. This feature is characteristic for CaM binding domains (O'Neil and DeGrado, 1990) but is not found in the majority of signal sequences. The HIV-1 envelope protein gp160 also has a signal sequence with an extended n-region that can potentially form a baa helix. As with p-Prl, we followed the fate of the p-gp160 signal sequence and found that a p-gp160 SPF is released into the cytosol and interacts with CaM. A synthetic p-gp160 SPF corresponding to the N-terminal 23 amino acid residues of the p-gp160 signal sequence has high affinity for CaM and efficiently inhibits Ca2+/CaM-dependent phosphodiesterase in vitro. Our results suggest that SPFs of distinct signal sequences may interfere with CaM functions and may act as regulatory peptides. Results Cleavage and processing of the p-Prl signal sequence We used a previously established system to follow the fate of the cleaved signal sequence of p-Prl (Lyko et al., 1995). A truncated mRNA coding for the 86 N-terminal amino acid residues of p-Prl was translated in the presence of rough microsomal membranes. Since truncated mRNAs lack a stop codon, termination of translation does not occur. Under these conditions p-Prl/86 chains are inserted into the translocation complexes of the microsomes and remain bound to the ribosome (Gilmore et al., 1991). Signal sequence cleavage does not occur because the p-Prl/86 chains are too short (Figure 1A, lane 1). To remove non-inserted p-Prl/86 chains, microsomes are then isolated, resuspended in cytosolic extracts and p-Prl/86 chains released from the ribosome by addition of puromycin. p-Prl/86 chains become translocated across the microsomal membrane and the signal sequence is cleaved. Finally, membranes are separated from the cytosol by centrifugation and both the membrane pellet and the cytosol (supernatant) are analysed by SDS–PAGE. Figure 1.Signal sequence cleavage, processing and release. (A) p-Prl*/86 chains were inserted into rough microsomes (lanes 1 and 2) and subsequently released from the ribosome by addition of puromycin. Incubation was continued for 1 (lanes 3 and 4) and 15 min (lanes 5 and 6). Before application to SDS–polyacrylamide gels membranes (pellet, P) were separated from the cytosol (supernatant, S) by centrifugation. SP indicates the cleaved signal sequence, SPF the signal peptide fragment. (B) Outline of the p-Prl signal sequence and the p-Prl SPF. The h-region of the signal sequence is shaded. Italic letters in the p-Prl SPF indicate that the C-terminal end of the fragment is estimated (see Lyko et al., 1995). Download figure Download PowerPoint When p-Prl/86 chains were released from the ribosome with puromycin and incubation was continued for 1 min, the signal sequence was cleaved by the signal peptidase and was found associated with the membrane (stage I; Figure 1A, lanes 3 and 4, and B). After longer incubation (15 min) the signal sequence was cleaved and processed further by an as yet unknown signal peptide peptidase and an SPF was found in the cytosol fraction (stage II; Figure 1A, lanes 5 and 6, and B). The same result was obtained when mRNA coding for a mutant p-Prl, p-Prl*, was used which contains additional methionines at positions 12 and 13 for better labelling of the SPF with [35S]methionine (Lyko et al., 1995). Membrane components interacting with the cleaved p-Prl signal sequence In order to probe the molecular environment of the signal sequence by site-specific photo-crosslinking, the photo-activatable amino acid L-4′-(3-[trifluoromethyl]-3H-diazirin-3-yl)phenylalanine [(Tmd)Phe; Figure 2A] was co-translationally incorporated into the p-Prl signal sequence instead of Val18 (see Figure 1B) to give p-Prl*T (for site-specific photo-crosslinking using (Tmd)Phe see Martoglio and Dobberstein, 1996, and references therein). p-Prl*T/86 chains were then used for membrane insertion and puromycin release as described above. Figure 2.Characterization of membrane components interacting with the cleaved p-Prl*T signal sequence. (A) Schematic illustration of the photo-activatable amino acid L-4′-(3-[trifluoromethyl]-3H-diazirin-3-yl)phenylalanine [(Tmd)Phe], which was site-specifically incorporated at position 18 of the p-Prl signal sequence (see also Figure 1B). (B) Photo-crosslinking of the cleaved p-Prl*T signal sequence to membrane components. p-Prl*T/86 chains were released from the ribosome by puromycin and incubated for 1 min at 22°C. Samples were then frozen in liquid nitrogen and subjected to UV light (lanes 2–6). Membranes (P) were then separated from the cytosol (S) by centrifugation and analysed for crosslink products (lanes 1–4) or immunoprecipitated with antibodies directed against the p-Prl SPF (lane 5), Prl (lane 6) and subunits of the signal peptidase complex (SPC21, lane 7, and SPC18, lane 8) respectively. The arrow indicates crosslinks to SPC21 and SPC18. Stars indicate the small crosslink product. (C) Identification of phospholipid as crosslink partner. Membranes were treated with phospholipase A2 (lane 3) after UV irradiation and separation from the cytosol. Samples were immunoprecipitated with anti-p-Prl SPF antibodies. The arrow indicates crosslinks to SPC 21 and SPC18, the star crosslinks to phospholipids. Download figure Download PowerPoint We first probed the molecular environment of the signal sequence at stage I, when the cleaved signal sequence is still associated with the membrane (see above). Crosslinking was induced with UV light 1 min after addition of puromycin. We found two major crosslink products with apparent molecular weights of ∼20 kDa and 4–5 kDa in the membrane fraction as revealed by SDS–PAGE (Figure 2B, lane 3, arrow and star). Immunoprecipitations with antibodies directed against the n-region of the p-Prl signal sequence and against prolactin (Prl) respectively showed that both crosslink products contain the cleaved signal sequence but not the Prl portion (Prl56; Figure 2B, lanes 5 and 6). Thus, the cleaved signal sequence (30 residues, ∼3–4 kDa) is crosslinked to components with estimated molecular weights of ∼17 and ∼1 kDa. Signal sequences are cleaved from the nascent precursor protein by the signal peptidase. We therefore assumed that the cleaved signal sequence is in contact with subunits of the pentameric signal peptidase complex (SPC) having molecular weights of 12, 18, 21, 22/23 and 25 kDa (Evans et al., 1986). Using antibodies against the four smaller SPC subunits we could immunoprecipitate the ∼20 kDa crosslink product with anti-SPC21 and to a minor extent also with anti-SPC18 antibodies (Figure 2B, lanes 7 and 8, arrow), but not with anti-SPC12 and anti-SPC22/23 antibodies (not shown). Sequence analysis of SPC21 and SPC18 has shown that these two subunits are putative serine proteases with homology to SEC11, an essential component of the signal peptidase complex in yeast (Böhni et al., 1988; Greenburg et al., 1989; Shelness and Blobel, 1990). We have previously reported that the signal sequence of p-Prl is in contact with lipid molecules when short p-Prl chains are inserted into the protein-conducting channel and the signal sequence is still attached to the precursor protein (Martoglio et al., 1995). Based on this finding and judged by the size of the small molecule (∼1 kDa) crosslinked to the cleaved signal sequence, we expected the 4–5 kDa crosslink product shown in Figure 2B (lanes 3 and 5, star) to be a lipid adduct. To test whether the low molecular weight crosslink partner is a phospholipid, we treated the sample after crosslinking with bee venom phospholipase A2 (Martoglio et al., 1995). Phospholipase A2 cleaves phospholipids at position C-2 into fatty acid and lysophospholipid. Because the amount of the 4–5 kDa crosslink product (Figure 2C, lane 2, star) was significantly reduced after treatment with phospholipase (Figure 2C, lane 3), we can conclude that a phospholipid is part of the respective crosslink product and hence that the cleaved signal sequence is also in contact with lipid molecules in the ER membrane. Interaction of the p-Prl SPF with a cytosolic protein We next probed the molecular environment of the signal sequence at stage II (see above). At this stage the p-Prl signal sequence has been cleaved and processed and an N-terminal SPF has been released into the cytosol (Figure 1A, lanes 5 and 6; Lyko et al., 1995). Crosslinking was now induced 15 min after addition of puromycin. In the sample subjected to UV light we found the SPF and a crosslink product with an apparent molecular weight of ∼20 kDa in the cytosol fraction (Figure 3A, lane 4). Immunoprecipitations with antibodies directed against the n-region of the p-Prl signal sequence (Figure 3A, lanes 5 and 6) and against Prl (Figure 3A, lanes 7 and 8) showed that the cytosolic crosslink product contains the SPF but not the Prl portion. Thus, the SPF is crosslinked to a component with an estimated molecular weight of 16–18 kDa (∼20 kDa minus ∼3 kDa from the SPF). Figure 3.The p-Prl*T SPF is crosslinked to a component present in cytosol. (A) Photo-crosslinking of the p-Prl*T SPF to a cytosolic protein. p-Prl*T/86 chains were released from the ribosome by puromycin in the presence of cytosol prepared from bovine brain (lanes 1–8) or GH3 pituitary cells (§, lanes 11 and 12). Cytosol was omitted in lanes 9 and 10. Samples were incubated for 15 min at 22°C and subjected to UV light (lanes 3–12) and membranes (P) separated from soluble components (S) by centrifugation. Samples were then analysed for crosslink products or immunoprecipitated with antibodies directed against the p-Prl SPF (lanes 5 and 6) and Prl (lanes 7 and 8) respectively. The major crosslink product is indicated by an arrow. (B) Photo-crosslinking of the p-Prl*T SPF in cytosol prepared from various sources. p-Prl*T/86 chains were released by puromycin as in (A) in the presence of cytosol prepared from bovine brain (bb, lanes 3 and 4), Mel Juso cells (mj, lanes 5 and 6) or wheatgerm extract (wg, lanes 7 and 8). Samples were further treated as in (A). The arrow indicates the major crosslink product in the cytosol fraction. Download figure Download PowerPoint The cytosol we used for the experiments shown in Figure 3 was prepared either from bovine brain (Figure 3A, lanes 1–8) or GH3 cells, a prolactin-synthesizing rat pituitary cell line (Figure 3A, lanes 11 and 12). When cytosol was omitted, no ∼20 kDa crosslink product was obtained in the ‘cytosol’ fraction (Figure 3A, lanes 9 and 10). We have also tested cytosol prepared from a human cell line (Mel Juso cells; Figure 3B, lanes 5 and 6) as well as wheatgerm extract (Figure 3B, lanes 7 and 8). As shown in Figure 3B, the ∼20 kDa crosslink product is always found when cytosol is present. This result suggests that the p-Prl SPF interacts with a cytosolic component uniformly present in higher eukaryotes. The p-Prl SPF interacts with calmodulin When p-Prl*T/86 chains were released from the ribosome with EDTA instead of puromycin, the cytosolic ∼20 kDa crosslink product was not found (Figure 4, lanes 1 and 2). This result suggests that release of the SPF and its binding to the cytosolic component depends on divalent cations. To test whether Ca2+ or Mg2+ is essential for binding, p-Prl*T/86 chains were released from the ribosome with puromycin in the presence of EGTA to chelate calcium ions. Again, no cytosolic crosslink product was observed (Figure 4, lanes 3 and 4). This suggests a calcium dependence of SPF binding to a cytosolic component. Figure 4.Identification of CaM as the crosslink partner. p-Prl*T/86 chains were released from the ribosome by EDTA (lanes 1 and 2) or puromycin (lanes 3–12) in the presence of cytosol (lanes 1–6), EGTA (lanes 3 and 4) or the CaM antagonist calmidazol (lanes 5 and 6). Cytosol was omitted in lanes 7–12 but purified CaM from bovine brain (lanes 7 and 8) or D.discoideum (§, lanes 9–12) were added instead. After UV irradiation membranes (P) were separated from soluble components (S) by centrifugation and analysed for crosslink products or immunoprecipitated with antibodies directed against D.discoideum CaM (lanes 11 and 12). Crosslinks to CaM are indicated by an arrow. Download figure Download PowerPoint The estimated molecular weight of the cytosolic component that is crosslinked with the p-Prl SPF is 16–18 kDa. Calmodulin (CaM) is a cytosolic calcium binding protein of ∼17 kDa and a central regulator of many kinases, phosphatases and transporters (Klee and Vanaman, 1982). To test whether the released p-Prl*T SPF interacts with CaM, the potent CaM antagonist calmidazol was added to the crosslinking assay. In the presence of calmidazol the cytosolic ∼20 kDa crosslink product was not observed (Figure 4, lanes 5 and 6), suggesting that calmidazol efficiently competes with the p-Prl SPF for CaM. As shown above, no ∼20 kDa crosslink product was obtained when cytosol was omitted (Figure 3A, lanes 9 and 10). When purified CaM (from bovine brain) and calcium were added, however, the ∼20 kDa crosslink product was obtained (Figure 4, lanes 7 and 8), suggesting that the p-Prl SPF is crosslinked to CaM. The ∼20 kDa crosslink product was also obtained when CaM prepared from Dictyostelium discoideum was added (Figure 4, lanes 9–12). CaM from D.discoideum was selected because a specific antiserum against this protein was available. With this antiserum we could immunoprecipitate the ∼20 kDa crosslink product and thus further characterize its identity (Figure 4, lanes 11 and 12). The p-Prl SPF is less efficiently released into the cytosol when factors are present that prevent an interaction with CaM (Figure 4, lanes 1–6) or when cytosol, and hence CaM, is absent (Figure 3A, lanes 9 and 10). This suggests that the interaction with CaM may facilitate the cytosolic localization of the amphipathic p-Prl SPF, which otherwise remains preferentially in the lipid bilayer. The efficiency of crosslinking between the p-Prl SPF in the cytosol and CaM was very high, up to 55% (estimated from Figure 3A, lanes 2 and 4), and indicates that the majority of p-Prl SPF was in contact with CaM. Because the amount of p-Prl SPF generated in our in vitro translation/crosslinking system is very low (20–100 fmol/20 μl reaction), the high crosslinking efficiency also indicates a high affinity of CaM for the p-Prl SPF. Furthermore, the high crosslinking efficiency is consistent with a tight interaction between the p-Prl SPF and CaM [for chemical properties of the carbene-generating (Tmd)Phe see Brunner, 1989]. Similar crosslinking efficiencies have been reported, for example, for the tight interaction between the signal sequence of a growing polypeptide chain and the 54 kDa subunit of the signal recognition particle during protein targeting (High et al., 1993b; Martoglio et al., 1995). Release of a SPF of HIV-1 p-gp160 into the cytosol and interaction with calmodulin The characteristic feature of a CaM binding domain is a stretch of 16–35 amino acid residues that can potentially form a basic amphiphilic α(baa)-helix (James et al., 1995). Such a stretch is predicted for the N-terminal portion of the p-Prl signal sequence. To see whether other signal sequences may also interact with CaM, we searched the signal sequences of mammalian and viral proteins listed in the SWISSPROT database for their potential to form a baa-helix. Most signal sequences are short (<20 amino acid residues) and, after cleavage by signal peptidase and processing by signal peptide peptidase, are not expected to bind to CaM. However, we found one more signal sequence comprising 30 amino acid residues and consensus features for CaM binding. The signal sequence of the HIV-1 envelope protein p-gp160 has all the features for a CaM binding peptide (Figure 5A). The n-region of the HIV-1 envelope protein p-gp160 signal sequence can potentially form a baa-helix and contains several tryptophan residues often found in CaM binding domains (Vorherr et al., 1990; James et al., 1995). Figure 5.Photo-crosslinking of the HIV-1 p-gp160T SPF to CaM. (A) Outline of the p-gp160 signal sequence. The shaded area indicates the h-region of the signal sequence. The N-terminal 23 residues are also illustrated in a helical wheel; hydrophobic residues are indicated as dark circles, basic residues as white circles. The arrow indicates the amino acid (G18) that was replaced with (Tmd)Phe for site-specific photo-crosslinking. (B) Release of the p-gp160 SPF into the cytosol. p-gp160/86 chains were inserted into rough microsomes (lanes 1 and 2) and subsequently released from the ribosome by addition of puromycin. Incubation was continued for 1 (lanes 3 and 4) or 15 min (lanes 5 and 6) and membranes (pellet, P) separated from the cytosol (supernatant, S) by centrifugation before SDS–PAGE. Lanes 4–6 show in vitro synthesized peptides corresponding to the 20, 25 and 30 N-terminal amino acid residues of p-gp160. The p-gp160 SPF released into the cytosol is indicated by a dot (lane 6). (C) Photo-crosslinking of the p-gp160T SPF to CaM. p-gp160T/86 chains were released from the ribosome by puromycin in the presence of cytosol prepared from Jurkat T cells (lanes 1–8, 14 and 15). Where indicated, calmidazol (lanes 5 and 6) or synthetic p-gp160 SPF corresponding to the 23 N-terminal residues of the p-gp160 signal sequence (lanes 7 and 8) was added in addition. Cytosol was omitted in lanes 9–13 and purified CaM from D.discoideum and Ca2+ were added in lanes 11–13. Samples were incubated for 15 min at 22°C and subjected to UV light (lanes 3–15) and membranes (P) separated from soluble components (S) by centrifugation. Membranes and the cytosol fraction of one sample were then treated with phospholipase A2 (lanes 14 and 15). Samples were finally analysed for crosslink products or immunoprecipitated with antibodies directed against D.discoideum CaM (lane 13) respectively. Crosslinks to CaM are indicated by an arrow, crosslinks to lipids by a star. Download figure Download PowerPoint With p-gp160 we performed analogous signal peptide release and crosslinking experiments as described above for p-Prl. Short p-gp160 chains (86 residues) were synthesized in vitro and inserted into the ER translocation sites of microsomal membranes (Figure 5B, lane 1). When p-gp160/86 chains were released from the ribosome by addition of puromycin and membranes were separated from the cytosol after 1 and 15 min incubation, a [35S]methionine-labelled peptide with an apparent molecular weight of 2–3 kDa appeared in the cytosol (Figure 5B, lanes 4 and 6). Because p-gp160/86 chains contain methionine residues only in the signal sequence, the released labelled peptide is either the cleaved signal sequence or a fragment thereof. To determine the approximate length of the peptide released into the cytosol, we synthesized marker peptides representing the entire p-gp160 signal sequence (30 amino acid residues) or N-terminal SPFs of 25 and 20 amino acid residues respectively. Comparative analysis of the peptides separated by SDS–PAGE revealed an estimated size of 20–25 amino acid residues, clearly smaller than the entire signal sequence (Figure 5B, lanes 6–9). This indicated that the released peptide is a fragment of the p-gp160 signal sequence and suggests that the p-gp160 signal sequence is rapidly processed. We could not detect a peptide corresponding to the entire signal sequence, as was the case for the signal sequence of p-Prl. The predicted processing site of the p-Prl signal peptide is between the two leucine clusters (Figure 1B) of its h-region (Lyko et al., 1995). Whether such a motif is required for signal sequence processing is not known. The h-region of the p-gp160 signal sequence also contains two clusters of amino acids with long hydrophobic side chains (-MLLGMLMI-) between which processing may occur (see Figure 5A). We next probed the molecular environment of the released p-gp160 SPF using site-specific photo-crosslinking. The photo-activatable amino acid (Tmd)Phe was co-translationally incorporated into the p-gp160 signal sequence instead of Gly18 (see Figure 5A) to give p-gp160T and p-gp160T/86 chains which were used for membrane insertion and puromycin release as described above. Crosslinking was induced with UV light 15 min after addition of puromycin. We found the SPF and a major crosslink product with an apparent molecular weight of ∼20 kDa in the cytosol fraction (Figure 5C, lane 4, arrow). This indicates that the peptide released into the cytosol contains (Tmd)Phe and hence must be derived from the p-gp160 signal sequence. The cytosol used for these experiments was prepared from Jurkat T cells. The same results were also obtained when cytosol prepared from bovine brain was used (not shown). To test whether the p-gp160T SPF is crosslinked to CaM, we released p-gp160T/86 chains from the ribosome in the presence of the CaM antagonist calmidazol. Furthermore, we released p-gp160T/86 chains when cytosol was omitted and when purified CaM and Ca2+ were added instead. The ∼20 kDa crosslink product was not observed in the presence of calmidazol (Figure 5C, lanes 5 and 6) or when cytosol was omitted (Figure 5C, lanes 9 and 10). The ∼20 kDa crosslink product was obtained, however, when calcium and purified CaM from D.discoideum (Figure 5C, lanes 11–12) or bovine brain (not shown) were present. In addition, we could immunoprecipitate the ∼20 kDa crosslink product with antiserum against CaM from D.discoideum (Figure 5C, lane 13). These results indicate that the p-gp160T SPF interacts with CaM. In analogy to p-Prl and based on the consensus for CaM binding, we expect that the released p-gp160T SPF comprises the N-teminal part of the signal sequence. Not all the p-gp160 SPF was released into the cytosol. After crosslinking a low molecular weight crosslink product appeared in the membrane fractions (Figure 5C, lane 3, star). This 4–5 kDa crosslink product was sensitive to phospholipase A2, indicating that it is a phospholipid adduct (Figure 5C, lane 14). Thus, some p-gp160T SPF remains in the membrane in contact with phospholipids. Characterization of the p-gp160 SPF–CaM complex CaM interacts with target proteins with affinity constants in the low nanomolar range (James et al., 1995). We determined the affinity constant for formation of the p-gp160 signal peptide–CaM complex by fluorometric titration using dansylated CaM (Anderson and Malencik, 1986) and a synthetic peptide corresponding to the 23 N-terminal residues of the p-gp160 signal sequence. This peptide could efficiently prevent crosslinking between the p-gp160T SPF and CaM in the signal peptide release and crosslinking experiment with p-gp160T/86 chains (Figure 5C, lanes 7 and 8). In the presence of calcium, dansyl-CaM showed a large increase in the fluorescence intensity upon binding of the peptide (Figure 6A). The Kd determined from two series of three titrations was 22 ± 5 nM and was derived from a non-linear curve fitting procedure. No fluorescence enhancement was observed when samples contained EGTA and no calcium (not shown). Figure 6.Characterization of the p-gp160 SPF–CaM complex. (A) Fluorescence titration of dansyl-CaM with p-gp160 SPF. Dansylated CaM [160 (♦) or 260 nM ] was titrated with a synthetic peptide corresponding to the 23 N-terminal residues of the p-gp160 signal sequence. The fluorescence intensity at 470 nm after excitation at 340 nm was recorded at each concentration of peptide. The fluorescence enhancement values were normalized for m" @default.
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• W2139833905 date "1997-11-15" @default.
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• W2139833905 title "Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin" @default.
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• W2139833905 doi "https://doi.org/10.1093/emboj/16.22.6636" @default.
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