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• W2034760458 abstract "Article15 February 1999free access The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules Michael M. Glombik Michael M. Glombik Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Andreas Krömer Andreas Krömer Search for more papers by this author Thorsten Salm Thorsten Salm Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Wieland B. Huttner Wieland B. Huttner Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Hans-Hermann Gerdes Corresponding Author Hans-Hermann Gerdes Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Michael M. Glombik Michael M. Glombik Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Andreas Krömer Andreas Krömer Search for more papers by this author Thorsten Salm Thorsten Salm Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Wieland B. Huttner Wieland B. Huttner Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Hans-Hermann Gerdes Corresponding Author Hans-Hermann Gerdes Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Michael M. Glombik1,‡, Andreas Krömer2,‡, Thorsten Salm1, Wieland B. Huttner1 and Hans-Hermann Gerdes 1 1Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany 2Institut für Klinische Pharmakologie Bobenheim, Richard-Wagner-Straße 20, D-67269 Grünstadt, Germany ‡M.M.Glombik and A.Krömer contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1059-1070https://doi.org/10.1093/emboj/18.4.1059 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein α1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3–AT–E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3–AT–E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN. Introduction During the biogenesis at the trans-Golgi network (TGN) of immature secretory granules (ISGs) and constitutive secretory vesicles (CVs), regulated secretory proteins are segregated from constitutive secretory proteins (Burgess and Kelly, 1987; Tooze and Huttner, 1990). Constitutive secretory proteins exit the TGN in CVs which continuously fuse with the plasma membrane (PM) to release their content. Regulated secretory proteins are sorted into ISGs which undergo maturation. During maturation, a subset of proteins is removed from ISGs in a process termed constitutive-like secretion (Arvan and Castle, 1998). The mature secretory granule is the storage compartment of regulated secretory proteins which are released from the cell after stimulation. Morphological (Tooze et al., 1987, 1989) and biochemical (Gerdes et al., 1989; Chanat and Huttner, 1991; Huttner et al., 1995) evidence suggests that a key step in the sorting of regulated secretory proteins at the level of the TGN is their selective aggregation. For chromogranin B (CgB) and secretogranin II (SgII) this aggregation depends on millimolar Ca2+ concentrations and on a mildly acidic pH, conditions which are present in the lumen of the TGN. Ca2+/pH-dependent aggregation of proteins is thought to be mediated by a structural feature composed of numerous acidic amino acids distributed over large parts of the polypeptide chain (Gerdes et al., 1989). Whether other motifs of regulated secretory proteins, comprised by short stretches of amino acids and often referred to as sorting signals, are also involved in sorting at the level of the TGN by a mechanism distinct from aggregation, is poorly understood. In the past, amino acid sequences of regulated secretory proteins have been found to affect storage in secretory granules of these proteins as determined by morphological analyses and stimulation-evoked secretion experiments (Stoller and Shields, 1989; Sevarino and Stork, 1991; Castle et al., 1992; Chevrier et al., 1993; Tam et al., 1993; Cool et al., 1995; Brechler et al., 1996; Varlamov and Fricker, 1996). However, it has not been shown whether the sequences identified were involved in protein sorting at the level of the TGN or in protein retention at the level of the ISG (Thiele et al., 1997). Furthermore, it is not known whether the identified sequences exert their effect on sorting via aggregation and/or via binding to membrane components in the TGN. The first indication for the existence of a specific sorting signal functioning at the level of the TGN was obtained for CgB, a regulated secretory protein of the granin family of proteins with widespread distribution in neuroendocrine cells. Near the N-terminus, CgB contains a disulfide-bonded loop which is highly conserved across species, and between CgB and chromogranin A (CgA) (Huttner et al., 1995), another member of the granin family. This loop motif is encoded by a separate exon, exon 3, for both CgB and CgA. Two lines of evidence show that the loop structure is necessary for sorting of CgB to the regulated pathway of protein secretion. First, reductive cleavage of the disulfide bond led to constitutive secretion of CgB (Chanat et al., 1993) but did not interfere with its aggregative properties in the TGN (Chanat et al., 1994). Secondly, vaccinia-based expression of a loop-deleted form of CgB, i.e. expression of the mutant in the absence of endogenous granin synthesis by infection with a recombinant virus, revealed that the 22 amino acids comprising the loop are necessary for sorting of CgB from the TGN to ISGs (Kroemer et al., 1998). The finding that the loop-deleted mutant of CgB is sorted to ISGs in the presence but not in the absence of full-length CgB highlighted the function of aggregative processes at the level of the TGN during sorting to the regulated pathway. Here we analyse whether the disulfide-bonded loop of CgB is sufficient to direct secretory proteins into ISGs and whether it promotes aggregation and/or membrane binding in the TGN. We generated fusion proteins of the loop motif and α1-antitrypsin, a constitutive secretory protein, and analysed their membrane binding, sorting and storage after transient transfection in the neuroendocrine cell line PC12. Results Expression of recombinant proteins CgB of different species shows a striking conservation of amino acids (aa) 1–43, of which aa 1–12 are encoded by the 3′ end of exon 2, and aa 13–43 by exon 3 (Pohl et al., 1990; Huttner et al., 1995). To analyse whether the N–terminal 43 aa of CgB (Figure 1A) contain sufficient sorting information to direct a constitutive protein to the regulated pathway of protein secretion, we fused this particular region to the constitutive secretory protein α1-antitrypsin. Two series of reporter constructs were produced, one employing the complete N-terminal sequence 1–43 of rat CgB referred to as E2/3, and one with aa 10–42 referred to as E3, the latter almost entirely encoded by exon 3. E2/3 and E3 contain 22 aa which form a disulfide-bonded loop (Figure 1A; Benedum et al., 1987). Figure 1.Schematic representation of reporter constructs. (A) N-terminal aa 1–43 of mature rat CgB. Grey box, sequence encoded by exon 3. The disulfide bond in this sequence is indicated by a bracket. The two double arrows indicate the sequence tested in reporter constructs (E2/3, E3). Note that E3 refers to aa 10–12 encoded by the end of exon 2 and to aa 13–43 encoded by exon 3. Numbers indicate position of amino acids in mature rat CgB. (B) Different recombinant proteins tested in this study. The constitutive reporter molecule AT was fused to the C-terminus of full length rat CgB (CgB–AT). E2/3 and E3 were inserted both at the N-terminus of AT (E2/3–AT and E3–AT, respectively). E2/3 was fused to the C-terminus of AT (AT–E2/3). E2/3 or E3 were inserted at the N- and C-terminus of AT (E2/3–AT–E2/3 and E3–AT–E3, respectively). Download figure Download PowerPoint We used a modified α1-antitrypsin (AT) containing a tyrosine sulfation site at the C-terminus (Leitinger et al., 1994). AT was shown to be efficiently secreted by PC12 cells (Kroemer et al., 1998). The tyrosine sulfation site allows [35S]sulfate labelling of the reporter fusion proteins specifically in the TGN, the compartment in which the initial segregation of regulated and constitutively secretory proteins takes place (Kelly, 1985; Tooze and Huttner, 1990). CgB sequences containing the disulfide-bonded loop, i.e. E2/3 or E3, were inserted three amino acids downstream of the signal peptidase cleavage site of AT and/or were fused to the C-terminus of AT (Figure 1B, see Materials and methods). Chimeric molecules with one loop, either at the N- or C-terminus, or with two loops, one at each terminus, were generated. Two controls, AT without rat CgB sequences and full-length CgB fused to the N-terminus of AT, were employed. Secretion kinetics of recombinant proteins We analysed the secretion of recombinant proteins in PC12 cells over a time period of 3 h. Transiently transfected cells were pulse-labelled for 5 min with [35S]sulfate. At the end of various chase times the labelled recombinant protein was quantitated in cell lysates and media after immunoprecipitation with an antiserum against AT followed by SDS–PAGE. During the first hour of chase the recombinant proteins were rapidly secreted (Figure 2). After 90 min of chase secretion reached a plateau (Figure 2, broken line). Notably, the plateau for each recombinant protein was at a different level indicating a different degree of secretion. Endogenous SgII, which is known to be sorted almost exclusively into ISGs upon exit from the TGN (Tooze and Huttner, 1990), exhibited only little secretion during the first 90 min. Notably, the t1/2 of secretion of all recombinant proteins was ∼20 min. This value is very similar to the t1/2 ∼17 min observed for AT after transient expression in Cos-7 cells (Leitinger et al., 1994) and is characteristic of constitutive protein secretion. Most likely two main components contribute to the secretion profile of the recombinant proteins; the portion released into the medium during the first 90 min reflects constitutive secretion in CVs, whereas the portion stored intracellularly beyond this time point implies storage in secretory granules. Figure 2.Secretion kinetics of recombinant proteins. PC12 cells transiently transfected with AT, E3–AT, E2/3–AT–E2/3 or CgB–AT were pulse-labelled with [35S]sulfate for 5 min. Equal aliquots of cell lysates and media collected at the end of the indicated chase times were analysed, either after immunoprecipitation (for recombinant proteins) or directly (endogenous SgII), by SDS–PAGE and phosphoimaging. Secretion values for each time point were calculated as percent of total (sum of cell lysate and medium). For AT, E3–AT, CgB–AT and SgII single experiments and for E2/3–AT–E2/3 the mean of two experiments are shown. The broken line indicates the endpoint of constitutive protein secretion. Download figure Download PowerPoint AT containing two loop motifs is significantly retained in PC12 cells Next, we quantitated the amount of all pulse-labelled recombinant proteins retained in PC12 cells after 90 min of chase. The relative amount stored intracellularly at this timepoint is referred to as storage efficiency (Seff). All recombinant proteins were detected as single bands of the expected mol. wt in cell lysates and in the corresponding media (Figure 3A). The total amounts of [35S]sulfate-labelled proteins with and without chase were compared and were found to be slightly increased after chase (not shown). This finding has been observed previously (Baeuerle and Huttner, 1987) and shows that no degradation of [35S]sulfate-labelled proteins occurred during the chase. Figure 3.Storage efficiency (Seff) of recombinant proteins after transient transfection. (A) PC12 cells transfected with the recombinant proteins (each on a separate dish) were pulse-labelled for 5 min with [35S]sulfate and chased for 90 min. Equal aliquots of cell lysates and media collected at the end of the chase were analysed for recombinant proteins by immunoprecipitation and SDS–PAGE followed by phosphoimaging. Signals for the indicated recombinant proteins in cell lysates (C) and media (M) of a single experiment are shown. The mol. wt of recombinant proteins (kDa) is indicated. (B) The graph shows the Seff of individual recombinant proteins calculated from several experiments. Error bars show standard deviation. Number (n) of experiments: AT, n = 9; CgB–AT, n = 7; E2/3–AT, n = 10; AT–E2/3, n = 12; E3–AT, n = 6; E2/3–AT–E2/3, n = 17; E3–AT–E3, n = 8. Download figure Download PowerPoint The basal level of storage in this system was determined by expression of AT alone. To determine the Seff of full-length CgB in this system, it was fused to AT (CgB–AT). For AT and CgB–AT, Seff of 7.9% ± 2.3 SD and 40.3% ± 6 SD, respectively, were determined (Figure 3B). The reduction in cellular storage of transfected CgB–AT compared with endogenous CgB (Seff ∼90%; Chanat et al., 1993), or exogenous human CgB (Seff ∼70%; Kroemer et al., 1998) most likely reflects steric hindrance in sorting of the fusion protein due to its AT moiety. When the E2/3 domain was present either at the N-terminus (E2/3–AT) or at the C-terminus (AT–E2/3) of AT, the Seff was found to be 14.2% ± 3.1 SD and 12.9% ± 3.3 SD, respectively (Figure 3B). Likewise, for E3, when inserted at the N-terminus of AT (E3–AT), a similarly low storage efficiency was obtained (Seff 12.5% ± 2.6 SD; Figure 3B). Importantly, Student's t-test analysis revealed that the Seff values of all recombinant proteins with one loop, though not as high as CgB–AT (Seff ∼40%), were significantly greater than that of AT (p ≤0.005). A more striking result emerged from the analysis of recombinant AT containing two disulfide-bonded loops per molecule, one located at the N-terminus and a second at the C-terminus. In the case of E2/3–AT–E2/3, the Seff of 33.3% ± 3.9 SD (Figure 3B) was significantly higher than that of AT fusion proteins with only one disulfide-bonded loop. Notably, E2/3–AT–E2/3 was stored to almost the same level as CgB–AT. The shorter fusion protein E3–AT–E3 was stored as high as E2/3–AT–E2/3 (Seff = 35.5% ± 3.9 SD; Figure 3B). This result clearly shows that amino acids 10–42 encoding the disulfide-bonded loop of CgB are sufficient to confer intracellular storage to AT when present at both ends of the reporter molecule. AT containing two disulfide-bonded loops is efficiently sorted to secretory granules The high Seff values of E2/3–AT–E2/3 and E3–AT–E3 suggested sorting of these proteins to the regulated pathway of protein secretion. This was investigated in three ways. First, we characterized the type of vesicle in which E2/3–AT–E2/3 left the TGN using a 5 min [35S]sulfate pulse followed by a 12 min chase. At this time point [35S]sulfate-labelled secretory proteins have left the TGN in secretory vesicles. CVs and ISGs were separated by sucrose gradient centrifugation as described previously (Tooze and Huttner, 1990). In addition to E2/3–AT–E2/3, AT and E2/3–AT, both with a low Seff (Figure 3B), were analysed in parallel. On the equilibrium gradient AT comigrated with heparan sulfate proteoglycan (hsPG), a marker for constitutive vesicles in PC12 cells (CV, Figure 4A). This result is consistent with the low Seff obtained for AT (Figure 3B) and confirms its secretion by the constitutive pathway. As expected from its slightly higher Seff, E2/3–AT largely comigrated with CVs (Figure 4B) and only a shoulder was present at the position of ISGs. In contrast, most of E2/3–AT–E2/3 comigrated with endogenous SgII, a marker protein for ISGs, whereas a minor portion comigrated with the marker for CVs, endogenous hsPG (Figure 4C). These data clearly show that the majority of E2/3–AT–E2/3 had entered the ISGs, whereas a minor portion exited the TGN in CVs. Determination of the sorting efficiency of E2/3–AT–E2/3 (see Materials and methods) revealed that at the level of the TGN ∼60% of E2/3–AT–E2/3 is sorted to ISGs. This sorting value is significantly higher than the storage value determined at the level of mature secretory granules after 90 min of chase (Figure 3B). Figure 4.E2/3–AT–E2/3 is sorted to immature secretory granules upon exit from the TGN. PC12 cells transfected with AT (A), E2/3–AT (B) or E2/3–AT–E2/3 (C) were pulse-labelled with [35S]sulfate for 5 min and chased for 12 min. Postnuclear supernatants were prepared and subjected to sequential velocity and equilibrium sucrose gradient centrifugation. Equal aliquots of gradient fractions were analysed, either after immunoprecipitation (AT, E2/3–AT, E2/3–AT–E2/3) or directly (hsPG, SgII), by SDS–PAGE and phosphoimaging. The graphs show the quantitation of the indicated [35S]sulfate-labelled proteins across the equilibrium sucrose gradient (fraction 1, top). The arrows indicate the peak of CVs and ISGs. Download figure Download PowerPoint Secondly, we performed double immunogold-electronmicroscopy on PC12 cells transiently transfected with E2/3–AT–E2/3. Two first antibodies were used, one against transfected AT and another specific for endogenous CgB. As shown in Figure 5, a specific labelling of E2/3–AT–E2/3 (large gold arrowhead) and CgB (small gold arrow) was obtained for dense-cored granules. Many granules showed colocalization of both proteins. When cells transiently transfected with AT were probed with an antibody against AT, no immunolabelling of dense core granules was detected (data not shown). The latter observation is consistent with the low storage efficiency of AT and its comigration with CVs on equilibrium gradients. Figure 5.Double immunogold electron microscopy showing colocalization of E2/3–AT–E2/3 and endogenous CgB in dense-cored secretory granules. Ultrathin cryosections prepared from PC12 cells transfected with E2/3–AT–E2/3 were double immunolabelled for E2/3–AT–E2/3 (large arrowhead) and endogenous CgB (small arrow). The bar corresponds to 300 nm. Download figure Download PowerPoint Thirdly, we tested for storage of recombinant AT containing two loops in secretory granules by performing depolarization-induced secretion experiments. Therefore PC12 cells, transfected with either E2/3–AT–E2/3 or E3–AT–E3, were [35S]sulfate pulse-labelled for 5 min followed by a chase of 90 min. Subsequently, the cells were stimulated by depolarization in a medium containing 55 mM potassium. The relative amount of [35S]sulfate-labelled proteins released into the medium after depolarization as compared with the cellular amount prior to stimulation, was quantified. As depicted in Figure 6, exogenous E2/3–AT–E2/3 and E3–AT–E3 were released to 11.7% ± 3.7 SD and 10.6% ± 3.1 SD, respectively. Simultaneously, endogenous SgII was released to similar amounts, i.e. 12.8% ± 6.1 SD from cells transfected with E2/3–AT–E2/3 and 12.0% ± 3.1 SD from cells transfected with E3–AT–E3. Under control conditions (either 5 mM KCl/2.2 mM CaCl2 or 55 mM KCl/10 mM MgCl2/no CaCl2), secretion of all three proteins was significantly lower. Consistent with the low Seff, a stimulated release of AT was not detected under the same experimental conditons (not shown). Taken together, these data show that E2/3–AT–E2/3 and E3–AT–E3 were sorted to ISGs upon exit from the TGN and stored in secretory granules which undergo a calcium-dependent stimulated secretion. Figure 6.Depolarization-induced, calcium-dependent secretion of E2/3–AT–E2/3 and E3–AT–E3. PC12 cells transfected with E2/3–AT–E2/3 (top) or E3–AT–E3 (bottom) were pulse-labelled for 10 min with [35S]sulfate and chased for 90 min. Cells were then incubated for 10 min as indicated. Equal aliquots of cells and media were analysed either directly for endogenous SgII, or by immunoprecipitation for transfected proteins, followed by SDS–PAGE and phosphoimaging. The graphs show the quantitation of released [35S]sulfate-labelled proteins during the 10 min incubation. Each value is expressed as percentage of total (sum of cells plus medium). Error bars show standard deviation of three independent experiments. Download figure Download PowerPoint Disulfide bond formation is necessary for efficient sorting of E2/3–AT–E2/3 Previously it was shown that reductive cleavage of the disulfide bond stabilizing the loop in CgB upon dithiothreitol (DTT)-treatment in vivo leads to missorting of CgB (Chanat et al., 1993). At the same time endogenous SgII, lacking cysteines, is efficiently sorted to secretory granules. We used this approach to analyse whether disulfide bond formation is important for the sorting of E2/3–AT–E2/3. The Seff of E2/3–AT–E2/3 was determined after pulse–chase labelling with [35S]sulfate in the absence and presence of 2 mM DTT. As a control, AT of transfected PC12 cells was analysed in parallel. Figure 7 shows that storage of E2/3–AT–E2/3 dropped significantly from 33.3% ± 3.9 SD in the absence of DTT to 7.0% ± 1.6 SD in the presence of DTT. Importantly, this value is very similar to that obtained for AT (Seff = 7.9% ± 2.3 SD in the absence of DTT; Seff = 9.4% ± 0.1 SD in the presence of DTT). This Seff reflects the background level of the constitutive reporter (see also Figure 3). In contrast, endogenous SgII with a Seff of 91.7% ± 3.6 SD in the absence of DTT was still stored efficiently in DTT-treated transfected PC12 cells (Seff = 68.8% ± 2.9 SD, Figure 7), as found previously by Chanat et al. (1993) for DTT-treated non-transfected PC12 cells. This indicates that the sorting machinery for the regulated pathway of secretion was still functioning in the presence of DTT. Therefore the lack of storage of E2/3–AT–E2/3 indicates that disulfide bond formation is necessary for sorting of E2/3–AT–E2/3 to the regulated pathway of protein secretion. Figure 7.Reductive cleavage of the disulfide-bonded loop induces missorting of E2/3–AT–E2/3. PC12 cells transfected with E2/3–AT–E2/3 or AT were pulse labelled in the absence or presence of 2 mM DTT. After 90 min of chase, equal aliquots of cell lysates and media were analysed either directly for SgII or by immuno-precipitation for recombinant proteins, followed by SDS–PAGE and phosphoimaging. Seff was calculated for the proteins indicated from three independent experiments, except for AT in the presence of 2 mM DTT, for which the standard deviation of a duplicate experiment is shown. Error bars, standard deviation. Download figure Download PowerPoint The loop structure of CgB is stabilized by an intramolecular disulfide bond (Benedum et al., 1987). Because E2/3–AT–E2/3 contains the loop at both termini, we analysed whether intermolecular disulfide bonds were formed between E2/3–AT–E2/3 molecules leading to covalently linked oligomers. These oligomers may mimic aggregates which could hypothetically result in their sorting to ISGs. To investigate this possibility we labelled E2/3–AT–E2/3, and as a control AT, which lacks cysteine-containing loops, in the TGN by a 5 min [35S]sulfate pulse. Subsequently the mol. wts of the 35S-labelled forms were analysed by SDS–PAGE under reducing and non-reducing conditions. Figure 8 shows that the mobility of neither AT nor E2/3–AT–E2/3 is affected by the presence or absence of β-mercaptoethanol. Note that AT and E2/3–AT–E2/3 were detected as doublets of lower (Figure 8, asterisk) and higher (Figure 8, arrowhead) mol. wt bands. In each case the lower band reflects incomplete sialylation (Leitinger et al., 1994) and is no longer seen after 5 min (Leitinger et al., 1994) or 90 min of chase (Figure 3). In conclusion, no intermolecular disulfide bonds had been formed between E2/3–AT–E2/3 molecules during biosynthesis and secretory transport to the TGN. Formation of ‘aberrant’ intramolecular disulfide bonds, i.e. between N- and C-terminal cysteines, we regard as most unlikely because E2/3–AT–E2/3 underwent [35S]sulfation in the TGN, showing that it passed the quality control mechanisms for misfolded proteins of the endoplasmic reticulum. Figure 8.E2/3–AT–E2/3 molecules do not form intermolecular disulfide bonds. PC12 cells transfected with AT or E2/3–AT–E2/3 were pulse-labelled for 5 min with [35S]sulfate. Cell lysates were analysed for recombinant proteins by immunoprecipitation followed by SDS–PAGE in the absence (−) or presence (+) of β-mercaptoethanol (β-ME), and phosphoimaging. Note the same mobility of AT or E2/3-AT-E2/3 under reducing and non-reducing conditions. In addition to the higher mol. wt form of labelled AT and E2/3–AT–E2/3 (arrowheads), which is the only form when pulse-labelling is followed by a chase (Figure 3A), a lower mol. wt form is visible for each protein (asterisks). This form reflects a different degree of sialylation. Download figure Download PowerPoint The disulfide-bonded loop does not promote Ca2+/low pH aggregation CgB has been shown to aggregate selectively in the presence of millimolar Ca2+ and mildly acidic pH, conditions mimicking the luminal milieu of the TGN (Chanat and Huttner, 1991). From this it has been proposed that selective aggregation of CgB in the TGN is an important step for its sorting to ISGs (Chanat and Huttner, 1991). To test whether the disulfide-bonded loop of CgB is involved in Ca2+/pH-mediated aggregation, we analysed in isolated TGN vesicles (Chanat and Huttner, 1991) the aggregative properties of AT bearing two loops. PC12 cells transfected with E2/3–AT–E2/3 were pulse-labelled for 5 min with [35S]sulfate. After subcellular fractionation TGN vesicle preparations were perforated with 0.1% saponin under aggregative (10 mM Ca2+ at pH 6.4) or non-aggregative conditions (absence of Ca2+ at pH 7.4). They were sedimented and supernatants and pellets were analysed for recombinant E2/3–AT–E2/3 and endogenous CgB. Under aggregative conditions 94.9% of full-length CgB and its fragments were obtained in the pellet, whereas under non-aggregative conditions endogenous CgB appeared almost completely in the supernatant and only 4.9% were found in the pellet (Figure 9). However, E2/3–AT–E2/3 was only detectable in the supernatant even in the presence of Ca2+/pH 6.4 (Figure 9). Thus, the disulfide-bonded loop does not confer the Ca2+/pH-dependent aggregative properties to the reporter and probably functions independently of Ca2+ and mildly acidic pH. Figure 9.E2/3–AT–E2/3 does not undergo Ca2+/pH-dependent aggregation. PC12 cells transfected with E2/3–AT–E2/3 were pulse-labelled for 5 min with [35S]sulfate. TGN vesicles prepared by velocity gradient centrifugation were perforated in a 0.1% saponin buffer, either under non-aggregative conditions (NA, absence of Ca2+ at pH 7.4), or under aggregative conditions (A, presence of 10 mM Ca2+ at pH 6.4.), and separated into supernatant (S) and pellet (P). Supernatants and pellets were divided into two equal aliquots. One aliquot of each fraction was subjected to immunoprecipitation for E2/3–AT–E2/3, the other for endogenous CgB. Immunoprecipitates were analysed by SDS–PAGE and phosphoimaging. Arrowhead, full-length endogenous CgB; brackets, degradation products of endogenous CgB; arrow, E2/3–AT–E2/3. Download figure Download PowerPoint The disulfide-bonded loop of CgB promotes binding to the TGN membrane of E2/3–AT–E2/3 To investigate whether E2/3–AT–E2/3 interacts with the membrane of the TGN we exploited sulfation to specifically label proteins in this compartment. A post-nuclear supernatant was prepared from transfected PC12 cells directly after pulse-labelling for 5 min with [35S]sulfate. At this time point no labelled proteins have exited the TGN (Tooze and Huttner, 1990). After opening of TGN vesicles by treatment with 0.03% saponin (Chanat and Huttner, 1991) followed by sonication, membranes were pelleted by centrifugation and the amount of [35S]sulfate-labelled E2/3–AT–E2/3 in the membrane fraction compared with the supernatant was determined and is referred to as binding efficiency. In parallel, membrane binding of tr" @default.
• W2034760458 created "2016-06-24" @default.
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• W2034760458 date "1999-02-15" @default.
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• W2034760458 title "The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules" @default.
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