Abstract
22 min readArticle Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV. https://doi.org/10.7554/eLife.26067.001 Introduction Proteins destined for the secretory pathway are translated and translocated into the endoplasmic reticulum (ER), which provides a specialized environment for their folding, disulfide bond formation, and N-linked glycosylation. Targeting of soluble and type-I transmembrane proteins to the ER is mediated via cleavable signal peptides, near-N-terminal hydrophobic stretches of 14–50 amino acids that are recognized by SRP (von Heijne, 1985; Kurzchalia et al., 1986; Lütcke et al., 1992; Walter and Blobel, 1981; Blobel and Dobberstein, 1975; Hegde and Bernstein, 2006). Cleavable signal peptides are variable in sequence but share characteristics of an N-terminal region with typically 0–2 basic residues, a membrane-spanning hydrophobic α-helix (H) region, and a C-terminal region that often contains a signal-peptide cleavage site (von Heijne, 1983; von Heijne, 1984). Signal-peptide cleavage is mediated by the signal-peptidase complex, which, like oligosaccharyl transferase, associates with the translocon (Görlich et al., 1992; Gilmore, 1993). In a second cleavage step signal peptides are cleared from the ER membrane by signal-peptide peptidase, an intramembrane rhomboid-like protease (Weihofen et al., 2002). Signal peptides are widely believed to be cleaved co-translationally (Blobel and Dobberstein, 1975; Jackson and Blobel, 1977; Martoglio and Dobberstein, 1998), but cleavage may well occur anywhere from early co-translational to late post-translational, depending on the protein. The minimal requirement for cleavage is the emergence of the cleavage site in the ER lumen, which translates to ~70 synthesized residues of a nascent polypeptide chain (Daniels et al., 2003; Kowarik et al., 2002; Hou et al., 2012), and its recognition by the signal-peptidase complex. Yet, examples such as preprolactin, influenza virus hemagglutinin (HA) and human cytomegalovirus US11 show that cleavage can follow initial folding and/or glycosylation and sometimes requires longer nascent-chain lengths (Daniels et al., 2003; Rutkowski et al., 2003; Rehm et al., 2001). The HIV-1 envelope glycoprotein gp160 represents an extreme case of late post-translational cleavage and the mechanism for delayed cleavage has been a long-standing problem (Li et al., 2000; Land et al., 2003; Li et al., 1996). The gp160 signal peptide acts as a membrane tether for at least 15 min after synthesis and requires at least some folding of the ectodomain for cleavage (Land et al., 2003). When proper folding is prevented by maintaining gp160 in a reduced state with DTT treatment or by blocking N-linked glycosylation using tunicamycin, the signal remains uncleaved and gp160 fails to exit the ER (Land et al., 2003). Gp160 begins as a transmembrane protein whose folding requires formation of conserved disulfide bonds, abundant glycosylation, proline isomerization, and trimerization before the protein leaves the ER (Land et al., 2003; Willey et al., 1988; Earl et al., 1990; Earl et al., 1991; Land and Braakman, 2001; van Anken et al., 2008). In the Golgi complex, gp160 glycans are modified and furin proteases cleave the trimeric glycoprotein into its two subunits, which stay non-covalently attached and are incorporated into new virions at the plasma membrane (Araújo and Almeida, 2013; Sundquist and Kräusslich, 2012). Gp160 is essential for virus entry as gp120 recognizes CD4, the HIV receptor on the cell surface, and one of the HIV coreceptors CCR5 and CXCR4, whereas gp41 mediates fusion with the target membrane (Blumenthal et al., 2012). The soluble subunit gp120 dominates gp160 folding and can fold and be secreted on its own, in the absence of gp41 (Land et al., 2003). Signal-peptide cleavage of gp120 is similarly delayed as for full-length gp160 and obeys the same rules (Land et al., 2003; van Anken et al., 2008). Therefore, for certain assays, we use gp120 interchangeably with gp160. Late cleavage of a signal peptide requires a two-component mechanism that prevents initial recognition of the cleavage site by the signal-peptidase complex and then enables cleavage at a later time. In this study, we identify the structural basis of delayed signal-peptide cleavage and propose a novel role for gp160 signal-peptide cleavage as a built-in quality control mechanism. Results Residues up- and downstream of the signal-peptide cleavage site prevent co-translational cleavage To examine whether the signal peptide directly impaired co-translational cleavage, we replaced the natural gp160 signal peptide (30 residues) with the unrelated signal peptides of influenza virus hemagglutinin (HA), cystatin (cys), Igκ, and two synthetic, optimal (opt), or suboptimal (sub) signal peptides (Barash et al., 2002). The timing of signal-peptide cleavage was assayed using a radioactive pulse-labeling approach (Land et al., 2003). Briefly, HeLa cells expressing wild-type or mutant gp120 were incubated with 35S-methionine and 35S-cysteine for 5 min and chased for the indicated times. Cells were transferred to 4˚C and iodoacetamide was added to prevent further formation and isomerization of disulfide bonds. Cells were lysed in Triton X-100 and the detergent lysates were immunoprecipitated with the polyclonal antibody 40336, which recognizes all forms of gp160. The immunoprecipitates were deglycosylated and subjected to reducing 7.5% SDS-PAGE to resolve signal-peptide-cleaved and uncleaved forms. After completion of synthesis, unprocessed deglycosylated gp120, with its signal peptide still attached (Ru) runs as a single band of ~70 kDa (Land et al., 2003) (Figure 1A). After a 60-min chase, a second band with increased mobility appears, representing signal-peptide-cleaved gp120 (Rc). In contrast, immediately after synthesis all gp120 constructs with non-native signal peptides already ran as a single band at the position of signal-peptide-cleaved gp120, indicating that the gp120 signal peptide is necessary for delayed signal cleavage. Figure 1 Download asset Open asset The interplay of gp120 and its natural signal peptide causes post-translational cleavage. (A) HeLa cells expressing wild-type gp120 (Wt) and gp120 with exogenous signal peptides, HA (HA), Ig κ (Ig κ), cystatin (Cys), Optimal (Opt), and Suboptimal (Sub), were radiolabeled for 10 min and chased for 1 hr or not (0'). After immunoprecipitation gp120 samples were deglycosylated and analyzed using reducing 7.5% SDS-PAGE. Gels were dried and exposed to Kodak-MR films. (B) As in (A) except Wt gp120, gp120 with the signal peptide of HA (HA-gp120), wild-type HA (HA-HA) and HA with the signal peptide of gp120 (gp120-HA) were labeled for 5 min and chased for indicated times. Ru: reduced gp120 with the signal peptide still attached; Rc: signal-peptide cleaved gp120; wt: wild-type gp120. Gels shown are representative of at least three independent experiments performed with fresh cells and transfections (biological replicates). https://doi.org/10.7554/eLife.26067.002 We next investigated whether the gp120 signal, alone, was sufficient for post-translational cleavage, and swapped the signal peptides of gp120 and the unrelated transmembrane protein, influenza virus HA. The HA-signal-peptide construct (HA-gp120) again was detected as co-translationally cleaved gp120 (Figure 1B). The gp120 signal peptide alone hence did not convert HA into a post-translationally cleaved protein. Both wild-type and signal-peptide-swapped HA (gp120-HA) ran as a single band on the gel. Therefore, features of both mature gp120 and its signal peptide are necessary for post-translational cleavage. To establish the minimal requirements of the gp160 sequence sufficient for post-translational cleavage, we designed a series of GFP constructs that contained the gp160 signal peptide and an increasing number of mature gp160 residues before GFP (Figure 2A, Figure 2—figure supplement 1A). GFP expression and localization were monitored by fluorescence microscopy (Figure 2—figure supplement 1B) and signal-peptide cleavage was assessed by immunoblot (Figure 2B). Figure 2 with 1 supplement see all Download asset Open asset Downstream residues of gp160 regulate signal-peptide cleavage. (A) Schematic of different GFP reporter constructs generated for imaging and immunoblot experiments. All reporters have the identical monomeric GFP-KDEL cassette. For ER GFP, the GFP-KDEL cassette is preceded by the bovine prolactin signal peptide (open box SP). The reporters with the HIV Env signal peptide (grey box SP) are fused to one or more amino acids of the mature gp120 domain followed by the GFP-KDEL cassette. (B) Western Blot analysis of control constructs (GFP-KDEL, ER-GFP) or gp160 signal peptide with 1-30 downstream residues (SP +1, …, 30). GFP-KDEL and SP +1 run lower on gel as they lack the additional residues downstream of the cleavage site. Ru: unprocessed molecules with signal peptide still attached; Rc: signal peptide-cleaved molecules. All images shown are representative of at least two independent experiments performed with fresh cells and transfections (biological replicates). https://doi.org/10.7554/eLife.26067.003 As expected, GFP attached to the gp160 signal peptide was correctly targeted to the ER and accumulated in the ER due to the engineered retrieval signal KDEL (Figure 2—figure supplement 1B SP+1, SP+10). Immunoblot analysis revealed a single band of GFP when one residue of mature gp160 sequence linked the signal peptide and GFP (SP+1), which migrated at the same position as control, GFP-KDEL (Figure 2B). Addition of four more residues of mature gp160 (SP+5) delayed the processing and resulted in signal-cleaved and uncleaved GFP species. Processing decreased even more with additional gp160 residues and the inclusion of ~10 downstream residues turned the signal peptide into a predominantly uncleaved signal anchor. We examined whether the population of cleaved and uncleaved molecules in steady state represented distinct populations or a low rate of cleavage. Pulse-chase experiments showed that cleavage only occurred during the pulse-labeling period and not anymore after synthesis (Figure 2—figure supplement 1C and D), demonstrating that signal-peptide cleavage of the GFP-reporter constructs was not progressive. We concluded that gp160 residues downstream of the signal cleavage site, in combination with the signal peptide itself, were sufficient to modulate cleavage efficiency of the gp160 signal peptide. Post-translational cleavage is conserved across HIV-1 subtypes Studies to date have primarily focused on the efficiency of signal-peptide cleavage of two closely-related isolates of HIV-1 Env, HXB2 (Li et al., 2000) and LAI (Land et al., 2003), which differ by only a single amino acid in the signal peptide (M24 in HXB2 and I24 in LAI) and 25 residues in gp120. To establish whether post-translational signal-peptide cleavage is conserved between different HIV-1 subtypes we examined the consensus sequence for each subtype and compared them to the sequence of subtype B strain HXB2 (Figure 3A). The amino acids responsible for the switch from signal peptide to signal anchor (Figure 2AB, +4 to +10) were shown to be highly conserved across all subtypes. Indeed, an alignment of more than 4100 sequences shows 99–100% conservation of these amino acids, with the exception of L34 which was 89% conserved (Supplementary file 1). As the residues immediately before and after the cleavage site are involved in recognition by the signal-peptidase complex (von Heijne, 1983; Choo and Ranganathan, 2008), we also examined the residues from positions −5 to +3 relative to the cleavage site. While the precise amino-acid conservation varied (16% for K33% to 87% for C28), amino-acid differences between sequences largely conserved the character of the amino acid in question (Supplementary file 1). Figure 3 Download asset Open asset Env posttranslational signal-peptide cleavage is conserved between subtypes. (A) Alignment of the signal peptide of HIV-1 Env reference strain HXB2 and the consensus sequences for subtypes A-E (www.hiv.lanl.gov). (B) Sequence alignment of HXB2 and subtype C strain 93IN101. Residues underlined are conserved between the two strains. (C) Western Blot analysis of HXB2 and 93IN101 SP +1 GFP and SP +10 GFP reporters. Blot shown is representative of two independent experiments performed with fresh cells and transfections (biological replicates). https://doi.org/10.7554/eLife.26067.005 The subtype-C consensus sequence demonstrated the greatest deviation from the HXB2 sequence in and around the cleavage site (Figure 3A). We therefore chose a subtype-C isolate with poor sequence conservation (93IN101), to compare signal-peptide cleavage with HXB2, using the GFP reporter system. Despite the low sequence similarity between the two isolates (Figure 3B), results for both 93IN101 SP+1 and SP+10 were comparable to those of HXB2, with efficient processing of SP+1 and delayed processing of SP+10. Notably the C isolate had a different ratio of cleaved and uncleaved, consistent with its slightly more efficient processing. Nevertheless, we concluded that post-translational signal-peptide cleavage is a conserved phenomenon across HIV-1 subtypes. The signal peptide acts as a signal anchor before cleavage We asked where the uncleaved signal peptide resides for the unprocessed protein. We hypothesized that the uncleaved signal peptide was either trapped in the Sec61 translocon, where signal peptidase is thought to reside primarily (Gilmore, 1993) or that the uncleaved protein was released laterally into the ER membrane where the uncleaved signal peptide may act as a transient signal anchor. To distinguish between these possibilities, we performed two different complementary assays. First, we assessed the mobility of several constructs in live cells using photobleaching analyses. A signal anchor should behave like a single-pass transmembrane protein, with a characteristic diffusion coefficient, while a protein trapped in the Sec61 translocon should exhibit very low mobility, as has been reported for the translocon (Nikonov et al., 2002). We compared diffusion rates of different GFP constructs determined from fluorescence recovery after photobleaching (FRAP) measurements (Figure 4A and B). Diffusion coefficients (D) differ from fast soluble proteins in the ER lumen (D = 8–12 µm/s2), to slower transmembrane proteins (D = 0.3–1 µm/s2), and very slow proteins attached to the translocon/ribosome complex (D = 0.04 µm/s2) (Nikonov et al., 2002). Signal-peptide-cleaved GFP (Figure 4B, SP+1) diffused rapidly (D = 9.6 ± 2.7 µm/s2), comparable with the ER-GFP control (D = 10.2 ± 1.6 µm/s2) and indicative of a soluble protein. Addition of ten gp160 residues downstream of the cleavage site to GFP (SP+10) slowed signal-peptide cleavage dramatically and resulted in a much lower D (D = 1.7 ± 0.6 µm/s2). The value was too high for a membrane protein, much too fast for a translocon protein, but much slower than for a soluble protein. The immunoblot data suggested that a mixed population is present in cells and our data are consistent with membrane and soluble species contributing to the diffusion coefficient. To validate membrane localization and to rule out that uncleaved SP+10 GFP was retained in the ER through trapping in the translocon, we used a carbonate-extraction approach, which releases translocon-bound clients into solution (Görlich et al., 1992) and retain integral membrane proteins in the pellet fraction (Figure 4C). Signal-cleaved SP+10 was fully soluble, while uncleaved SP+10 GFP was only found in the pellet fraction, indicating that it was fully integrated in the membrane and not in the translocon protein channel. Thus, the presence of a minimum of 5 to 10 gp120 amino acids downstream of the cleavage site prevented co-translational signal-peptide cleavage an anchored GFP to the membrane through this signal peptide. Figure 4 Download asset Open asset The uncleaved signal peptide acts as a signal anchor. (A) FRAP analysis of gp160 signal-peptide constructs as in Figure 2. Cos-7 cells expressing SP+1 and SP+10 GFP reporters were subjected to FRAP analysis. A small region of interest (white outlined box) was photobleached with intense laser light and imaged with low laser light to visually (A) and quantitatively (B) compare mobilities and fluorescence-intensity recovery rates. (A) Both reporters are mobile and unbleached reporters diffuse into the photobleached region of interest. Scale bar = 10 μm. (B) Plot of representative fluorescence recoveries into the photobleach region of interest reveals that SP+10 is slower to recover. Number of cells analyzed, diffusion constants and, statistical values can be found in Figure 4—source data 1. (C) Western Blot analysis of pellet and supernatant (sup) fractions from a carbonate extraction of cells expressing SP+10. Split band for GFP in Sup is likely due to fragmentation (Wei et al., 2015). Blots in panel C are representative of at least two independent experiments (biological replicates). https://doi.org/10.7554/eLife.26067.006 The signal-peptide-cleavage site is at the membrane-lumen interface To establish whether the signal-peptide-cleavage site is exposed outside the ER membrane in wild-type gp120, we determined which residue is the first protruding into the ER lumen by in-vitro translation of a construct composed of 88 N-terminal gp120 residues (including the signal peptide) coupled to the P2 domain of leader peptidase (Lep) in the presence of rough microsomes as a source of ER membranes (Nilsson and von Heijne, 1993) (Figure 5A). The construct contains two glycosylation sites. The first, within Lep, was readily glycosylated and served as a translocation control. The second glycosylation site was moved through positions 39–47 to determine the position at which it became glycosylated. N-linked glycosylation requires a minimal distance from the ER membrane of ~11–12 residues and therefore, glycosylation can be used as a ‘molecular ruler’ of lumenal exposure (Nilsson and von Heijne, 1993; Bañó-Polo et al., 2011). Figure 5 Download asset Open asset The signal-peptide cleavage site is buried in the membrane. (A) Cartoon of the first 89 residues of gp160 attached to the P2 domain of protein leader peptidase (Lep) at the C-terminus. A first glycosylation site in Lep was used as translocation control. The second site was introduced at positions 39–47 to determine at which position the distance to the membrane is sufficient for glycosylation. Arrow indicates site of signal-peptide cleavage. (B) Gp160-Lep constructs with glycosylation sites N39, N40, N41, and N47, were in-vitro translated in presence (+) or absence (-) of dog pancreas microsomes (RM). Gp160-Lep received a single (<) or double (<<) glycan, or remained unglycosylated/untranslocated (o). (C) Band intensities of B were quantified and percentage of second glycosylation product was plotted against position of the engineered glycosylation site. The data in panels B and C are representative of multiple independent experiments (biological replicates). https://doi.org/10.7554/eLife.26067.008 Positions 39 and 40 were poorly glycosylated and thus located close to the ER membrane (Figure 5B and C). Glycosylation of the second site sharply increased at position 41 and beyond, which thus is located 11–12 residues away from the membrane-water interface. This result strongly supports the interpretation that the cleavage site of gp160’s signal peptide is at the membrane-lumen interface, with T31 barely exposed in the ER lumen. The active site of signal peptidase is predicted to be 0.4–1.1 nm beyond the membrane surface (Liang et al., 2003). It therefore is likely that the shielding of the cleavage site by membrane phospholipids contributes to the delayed cleavage of the signal peptide. Secondary structure prevents co-translational signal-peptide cleavage Taken together, our results suggest a model in which the signal peptide and flanking gp120 sequence are sufficient to delay signal-peptide cleavage and that the cleavage site is poorly accessible. The signal peptide and flanking domain hence appear to regulate signal-peptide cleavage. To characterize the physical properties of the gp160 signal peptide, the sequence was analyzed with the online prediction tool SignalP (Bendtsen et al., 2004). A classical signal peptide, prolactin, exhibits a clear separation of N-, H-, and C-regions and a high signal-peptide probability in the hidden Markov model (Bendtsen et al., 2004) (Figure 6A). In contrast, gp160’s signal peptide contains a predicted hydrophobic H-region that overlaps substantially with the C-region containing the cleavage site (Figure 6A). This predicted structure was highly unusual, as analyses of numerous other cleaved signal peptides did not exhibit overlap of H and C regions (our unpublished data). The probabilities for the gp160 signal peptide to be a cleavable signal peptide or an uncleaved signal anchor were 0.628 and 0.358, respectively. Similar probabilities were obtained for gp160 sequences from five different subtypes. SignalP analysis further corroborated our biochemical findings that the downstream residues of the mature gp120 influence probability of signal-peptide cleavage. When we tested the impact of the mature HA sequence on the gp160 signal peptide, separation between the H- and C-region visibly improved and cleaved signal-peptide probability increased to a near perfect 0.936 (Figure 6A), consistent with our experimental findings (Figure 1B). Figure 6 with 1 supplement see all Download asset Open asset The C region of the gp160 signal peptide overlaps with the hydrophobic core region. (A) Signal-peptide prediction tool SignalP 3.0 (36) was used to assess signal peptides of prolactin, gp160, and gp160 signal peptide followed by HA (as used in Figure 1B). The characteristic N-terminal charged region (N), the hydrophobic membrane-spanning region (H), and the C-terminal region (C) containing the cleavage site were plotted. Vertical bars represent the first amino acid after the cleavage site. (B) TopPred1 (Goldman, Engelman, and Steitz scale), TopPred2 (Kyte and Doolittle scale), TMPred, TMHMM, HMMTOP, PHDHTM, and NetSurf Helix were used to predict the transmembrane domain of the gp160 signal peptide. The predicted transmembrane domains are represented by grey bars below the HXB2 sequence (residues 12–45). The cleavage site is marked by a vertical line. (C) Robetta (Kim et al., 2004) was used to predict the structure of the area around the signal-peptide cleavage site of wild-type gp160. Residues M26, A30 and T31 are shown as sticks. Alpha helices colored in grey. (D) SignalP prediction of gp160 M26P signal peptide. (E) as in (C) except M26P gp160 was used for the prediction. Structures shown are representative of the 5 predicted structures received from the Robetta server. https://doi.org/10.7554/eLife.26067.009 The SignalP predictions combined with our finding that T31 is at the lumen-membrane interface suggest that the membrane-spanning alpha helix of the gp160 signal peptide extends beyond position 31, allowing immersion into the membrane and occluding the cleavage site. Secondary structure is known to inhibit cleavage by proteases and could account for the initial resistance of the gp160 signal peptide to signal-peptidase activity (Fluhrer et al., 2012). Indeed, the majority of transmembrane-domain prediction algorithms suggested that residues 33 up to 44 of gp160 form an extended alpha helix (Figure 6B). De novo structure predictions using Robetta (Kim et al., 2004) also show the alpha helix overlapping with the cleavage site (Figure 6C). Together, these analyses suggest a model in which an extended α-helical structure around the cleavage site impairs accessibility to signal peptidase. Breaking secondary structure causes co-translational signal-peptide cleavage If helical structure around the cleavage site is prohibitive, disruption of the helix would be predicted to lead to co-translational signal-peptide cleavage of gp160. Importantly, a point mutation in the signal peptide should not directly impact mature gp160, whose sequence is unaltered. To break secondary structure, we introduced a proline in the signal peptide close to the cleavage site. Prolines are common in signal peptides but are absent in 99.9% of known HIV-1 gp160 sequences (Supplementary file 1). SignalP analysis revealed that mutation M26P increased signal-peptide cleavage probability from 0.628 to 0.928 and clearly separated H- and C-regions (Figure 6D). Similarly, de novo structure predictions also showed a break in alpha-helical structure around the cleavage site (Figure 6E, Figure 6—figure supplement 1). To study timing of signal-peptide removal and the potential impact on the folding and maturation of gp160, we analyzed the fate of M26P gp160 in our folding assay (Figure 7A). On a reducing gel (Cells R) wild-type gp160 with its signal peptide still attached (Ru) runs as a single band of ~100 kDa after synthesis (Land et al., 2003). From 15 min onward signal peptide-cleaved gp160 (Rc) appears just below Ru. Upon proper folding, trimerization, furin cleavage, and arrival at the plasma membrane, gp120 sheds from gp41 and is detected in the medium (Moore et al., 1990). Figure 7 Download asset Open asset M26P leads to co-translational signal-peptide cleavage. Experiments were done as in Figure 1. (A) HeLa cells expressing wild-type and M26P gp160 were radiolabeled for 10 min and chased for the indicated times. Samples were deglycosylated with endoH and subjected to reducing (Cells R) and non-reducing (Cells NR) 7.5% SDS-PAGE. Medium samples were reduced and not deglycosylated. Lane profiles depicting the folding-intermediate (NR) smear of wild-type and M26P gp160 were determined from autoradiographs. (B) As in A except that wild-type and M26P gp120 were used and samples were pulse labeled for 5 min in the presence of 5 mM DTT and chased in the absence of DTT. (C + D) HeLa cells expressing wild-type and M26P gp120 were pulse labeled as above and chased for either 0, 2, or 5 min (C) or 2 h (D). At the end of each time point, detergent cell lysates were proteolyzed with 5 or 100 µg/ml trypsin for exactly 15 min on ice. Proteolyzed samples were processed as in Figure 1 and analyzed by 15% SDS-PAGE. (E) HeLa cells expressing wild-type or M26P gp160 were pulse labeled and chased as above. Detergent cell lysates were immunoprecipitated in parallel with either polyclonal antibody 40336 or antibodies A32, D7324, or CD4-IgG. After immunoprecipitation, samples were processed as in Figure 1. (F) Quantifications of experiments from E. Values were normalized compared to immunoprecipitation by 40336. Statistics were calculated using an unpaired t-test with Welch’s correction. Exact p values can be found in Figure 7—source data 1. IT: folding intermediates; NT: native gp160; *: uncleaved unglycosylated M26P gp160 that had not targeted properly to the ER, likely due to its suboptimal signal sequence. Gels shown are representative of at least 3 independent experiments (biological replicates). https://doi.org/10.7554/eLife.26067.011 Immediately after synthesis M26P gp160 already ran primarily as a single band in the position of signal-peptide-cleaved gp160 (Figure 7A, Cells R). As predicted, cleavage occurred co-translationally. The overall signal of EndoH-sensitive cell-associated M26P gp160 and kinetics of gp120 shedding were comparable to wild-type type protein (Figure 7A). Even though M26P gp160 signal peptide was rapidly removed, the mutant took as long as wild-type gp160 to fold and exit the ER. We did not detect any difference in aggregation or degradation of wild-type versus M26P gp160. A second, faster-migrating band of M26P (asterisk) was identified as cytosolic and attributed to suboptimal targeting to the ER due to weaker SRP binding: it did not change between reducing and non-reducing gels (Figure 7A, asterisk), or when DTT was removed from cells (Figure 7B, asterisk), indicating that it did not form disulfide bonds, it still contained the signal peptide, but did not contain the ~25 GlcNAc m
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