Signal Sequence Mutations Research Articles

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

HIV-1 envelope glycan composition influences virus-host interaction

Abstract HIV-1 envelope (Env) is densely shrouded with oligomannose- and complex-type glycans. These glycans interact with lectins, a family of carbohydrate-recognizing proteins. We evaluated lectins for the ability to block infection of HIV-1 isolates that differ in their sensitivity to neutralizing antibodies. The antibody-sensitive tier 1 viruses were blocked by lectins more easily than the resistant tier 2 viruses. When tier 2 viruses were produced with glycosidase inhibitors to express homogenously oligomannose-type glycans, they became more sensitive to mannose-binding lectins (GNA, GRFT, CVN), suggesting that glycan homogeneity increases virus sensitivity to antibodies and lectins. Further, we tested the effect of glycan content on HIV-1 capture and transmission via DC-SIGN, a C-type lectin expressed by macrophages and dendritic cells. While the capture of virus enriched with high-mannose glycans was enhanced, transmission was diminished due to increased degradation. Under a biologically relevant setting, glycan composition was influenced by the Env signal sequence (SS), and SS mutations altered DC-SIGN-mediated virus transmission. The SS mutants also displayed altered sensitivity to inhibition by lectins GNA and GRFT. Mutations in gp120 did not have these effects. Env SS sequences and glycan compositions also vary among HIV-1 strains, leading to differences in their interactions with and sensitivity to lectins. These phenotypes were associated with the amounts of terminating Manα1–3 vs Manα1–2 residues on the Env oligosaccharides and their recognition by DC-SIGN, GNA, and GRFT. These data demonstrate the importance of the Env sugar contents for HIV-1 interactions with lectins expressed by the host immune cells.

Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability

SUMMARYCells have evolved quality control pathways to prevent the accumulation of improperly localized proteins, which are often toxic. One of these pathways, regulation of aberrant protein production (RAPP), recognizes aberrant secretory proteins during translation and degrades the associated mRNA. Here, we demonstrate endogenous RAPP substrates. Haploinsufficiency of the secretory protein progranulin (GRN) is associated with the neurodegenerative disease frontotemporal lobar degeneration (FTLD). Our results show FTLD-associated GRN mutations W7R and A9D disrupt co-translational interaction with a targeting factor, signal recognition particle (SRP). This triggers RAPP and initiates specific mRNA degradation. Conversely, wild-type GRN and the naturally occurring polymorphism V5L GRN are efficiently expressed and secreted. Thus, RAPP plays a role in the molecular pathology of A9D GRN and W7R GRN.

Escherichia coli SecG is required for residual export mediated by mutant signal sequences and for SecY-SecE complex stability.

Protein export to the bacterial periplasm is achieved by SecYEG, an inner membrane heterotrimer. SecY and SecE are encoded by essential genes, while SecG is not essential for growth under standard laboratory conditions. Using a quantitative and sensitive export assay, we show that SecG plays a critical role for the residual export mediated by mutant signal sequences; the magnitude of this effect is not proportional to the strength of the export defect. In contrast, export mediated by wild-type signal sequences is only barely retarded in the absence of SecG. When probed with mutant signal sequences, secG loss of function mutations display a phenotype opposite to that of prlA mutations in secY. The analysis of secG and prlA single and double mutant strains shows that the increased export conferred by several prlA alleles is enhanced in the absence of SecG. Several combinations of prlA alleles with a secG deletion cannot be easily constructed. This synthetic phenotype is conditional, indicating that cells can adapt to the presence of both alleles. The biochemical basis of this phenomenon is linked to the stability of the SecYE dimer in solubilized membranes. With prlA alleles that can be normally introduced in a secG deletion strain, SecG has only a limited effect on the stability of the SecYE dimer. With the other prlA alleles, the SecYE dimer can often be detected only in the presence of SecG. A possible role for the maintenance of SecG during evolution is proposed.

A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments

Bacterial microcompartments (MCPs) are protein-based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high-throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP-targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP-targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways.

Inefficient SRP Interaction with a Nascent Chain Triggers a mRNA Quality Control Pathway

Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).

Autoimmune lymphoproliferative syndrome due to FAS mutations outside the signal-transducing death domain: molecular mechanisms and clinical penetrance

PurposeAutoimmune lymphoproliferative syndrome is a disorder of lymphocyte apoptosis. Although FAS molecules bearing mutations in the signal-transducing intracellular death domain exhibit dominant-negative interference with FAS-mediated apoptosis, mechanisms for pathology of non-death domain FAS mutations causing autoimmune lymphoproliferative syndrome are poorly defined. MethodsRNA stability, protein expression, ligand binding, and ability to transmit apoptosis signals by anti-FAS antibody or FAS ligand were determined for a cohort of 39 patients with non-death domain autoimmune lymphoproliferative syndrome. Correlations between mutation type and disease penetrance were established in mutation-positive family members. ResultsFrameshifts or transcriptional stop mutations before exon 7 resulted in messenger RNA haploinsufficiency, whereas an amino-terminal signal sequence mutation and certain intracellular truncations prevented cell surface localization of FAS. All resulted in decreased FAS localization, inability to bind FAS ligand, and reduced FAS ligand-induced apoptosis. Extracellular missense mutations and in-frame deletions expressed defective FAS protein, failed to bind FAS ligand, and exhibited dominant-negative interference with FAS-mediated apoptosis. Mutation-positive relatives with haploinsufficient or extracellular mutations had lower penetrance of autoimmune lymphoproliferative syndrome clinical phenotypes than did relatives with death domain mutations. ConclusionWe have defined molecular mechanisms by which non-death domain FAS mutations result in reduced lymphocyte apoptosis, established a hierarchy of genotype-phenotype correlation among mutation-positive relatives of patients with autoimmune lymphoproliferative syndrome, and demonstrated that FAS haploinsufficiency can lead to autoimmune lymphoproliferative syndrome.Genet Med 2012:14(1):81–89

MicroRNA-155 Modulates Transforming Growth Factor-β Signaling In Chronic Lymphocytic Leukemia through Targeting of Casein Kinase γ Isoform 2

AbstractAbstract 3584Chronic lymphocytic leukemia (CLL) is typically characterized by defects in programmed cell death rather than alterations in cell cycle regulation. Transforming growth factor β (TGFβ), a ubiquitously expressed growth factor, regulates multiple normal cellular responses including proliferation, differentiation, migration and apoptosis. Loss of growth inhibition by TGFβ is thought to contribute to the development and progression of a variety of tumors including CLL (DeCoteau et al., PNAS 1997). Approximately 40% of patients contain mutations in the signal sequence of TGFβ receptor 1 (TBR-1) in the form of substitutions or deletions (Schiemann et al., Cancer Detect Prev 2004). In the wild type form, the signal sequence contains a nine alanine stretch, which if truncated has been shown to impair signaling through the receptor and specifically, a truncated, six alanine form is associated with increased cancer risk (Pasche et al., Cancer Res 1999). TGFβ signaling can regulate expression of micoRNAs (miRNA), which are ~22 nucleotide-long RNA gene regulators. Deregulated miRNA expression has been implicated in tumorigenesis, including CLL. Several miRNAs have been shown to be over-expressed in CLL as compared to normal B cells (Fulci et al., Blood 2007). This includes miR-155, which is part of a 13-miRNA signature that has prognostic implications, including a shorter need-for-treatment interval (Calin et al., N Engl J Med 2005). Interestingly, miR-155 has been shown to be upregulated by TGFβ in murine mammary gland cells (Kong et al., Mol Cell Biol 2008). The goals of our study are to investigate the link between TGFβ signaling and miR-155 in CLL and to determine how the interaction between the two may contribute to the pathogenesis of CLL. Here we show that miR-155 is in fact upregulated by TGFβ in mouse splenic B cells and in human peripheral blood B cells. In CLL, miR-155 expression inversely correlates with the proportion of CLL cells harboring signal sequence mutation in TBR-1, consistent with miR155 regulation by TGFβ in vivo. To understand the role of TGFβ-induced miR-155 in CLL pathobiology, identification of specific target genes in the context of this disease is essential. To this end, we compared the gene (cDNA) expression profile between CLL with high miR-155 vs. low miR-155 expression and identified putative miR-155 target genes by selecting those genes that are differentially expressed in SAM analysis with lower expression in the high miR-155 group, and which harbor predicted miR-155 binding sites in their 3' untranslated region (UTR). Based on this algorithm, we have identified casein kinase 1 gamma 2 (CSK1γ2) as a target for miR155 in CLL. CSK1γ2 is a negative modulator of the TGFβ signaling pathway by targeting the phosphorylated form of SMAD3 for degradation (Guo et al., Oncogene 2008). MiR-155 represses luciferase reporter gene expression by specific binding to the miR-155 site in the CSK1γ2 3'UTR. In addition, we found that CSK1γ2 itself is upregulated in B cells upon TGFβ stimulation, and treatment of human B cells with PNA miR-155 inhibitor (Fabani et al., Nucleic Acids Research 2010) further increases CSK1γ2 mRNA levels. Surprisingly, comparison of CSK1γ2 protein levels between CLLs with high or low miR-155 by Western blotting revealed higher CSK1γ2 protein expression despite lower CSK1γ2 mRNA levels, suggesting that miR-155 may enhance CSK1γ2 translation in CLL cells and implying an intriguing regulatory interaction between miR-155 and CSK1γ2. In summary, our data indicates that the variation of miR-155 seen in CLL is primarily a function of TGFβ signaling activity. Moreover, miR-155 is an important player in a complex auto-regulatory network in TGFβ signaling by fine-tuning the negative feedback mechanism on TGFβ signaling mediated by CSK1γ2. In CLL cells harboring TBR-1 with wild-type signal sequence, higher miR-155 levels may help modulate the TGFβ signaling activity to a level optimal for the survival or other pathobiological functions of CLL. Furthermore, since CLL cells are predominantly non-proliferating, our findings that miR-155 may enhance translation of CSK1γ2 provide support to the model of cell cycle dependence of microRNA functions (Vasudevan et al., Cell Cycle 2008). Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanisms Leading to Null-Protein Product from Retinoschisin (RS1) Signal-Sequence Mutants in X-Linked Retinoschisis (XLRS) Disease

Retinoschisin (RS1) is a cell-surface adhesion molecule expressed by photoreceptor and bipolar cells of the retina. The 24-kDa protein encodes two conserved sequence motifs: the initial signal sequence targets the protein for secretion while the larger discoidin domain is implicated in cell adhesion. RS1 helps to maintain the structural organization of the retinal cell layers and promotes visual signal transduction. RS1 gene mutations cause X-linked retinoschisis disease (XLRS) in males, characterized by early-onset central vision loss. We analyzed the biochemical consequences of several RS1 signal-sequence mutants (c.1A>T, c.35T>A, c.38T>C, and c.52G>A) found in our subjects. Expression analysis in COS-7 cells demonstrates that these mutations affect RS1 biosynthesis and result in an RS1 null phenotype by several different mechanisms. By comparison, discoidin-domain mutations generally lead to nonfunctional conformational variants that remain trapped inside the cell. XLRS disease has a broad heterogeneity in general, but subjects with the RS1 null-protein signal-sequence mutations are on the more severe end of the clinical phenotype. Results from the signal-sequence mutants are discussed in the context of the discoidin-domain mutations, clinical phenotypes, genotype-phenotype correlations, and implications for RS1 gene replacement therapy.

Genetic Suppressors and Recovery of Repressed Biochemical Memory

Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1–3). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories.

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

The cellular prion protein PrP(C) is synthesized as a family of four distinct forms. Of these, (Cyt)PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that (Cyt)PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G(0)/G(1) phase. The identification of this PrP isoform and its properties provides novel insight into PrP(C) physiological and pathological functions.

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys --> Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.

The Plug Domain of the SecY Protein Stabilizes the Closed State of the Translocation Channel and Maintains a Membrane Seal

Proteins are translocated across membranes through a channel that is formed by the prokaryotic SecY or eukaryotic Sec61 complex. The crystal structure of the SecY channel from M. jannaschii revealed a plug domain that appears to seal the channel in its closed state. However, the role of the plug remains unclear, particularly because plug deletion mutants in S. cerevisiae are functional. Here, we demonstrate that plug deletion mutants in E. coli SecY are also functional and even efficiently translocate proteins with defective or missing signal sequences. The crystal structures of equivalent plug deletions in SecY of M. jannaschii show that, although the overall structures are maintained, new plugs are formed. These lack many interactions that normally stabilize the closed channel, explaining why the channels can open for proteins with signal-sequence mutations. Our data show that the plug domain is required to maintain a closed state of the channel and suggest a mechanism for channel gating.

ER stress and apoptosis‐inducing mutations in carbonic anhydrase IV associated with retinitis pigmentosa (RP17) respond to chemical chaperones

We examined two human mutations in the carbonic anhydrase IV (CA IV) gene, which are associated with RP17, an autosomal dominant form of retinitis pigmentosa. CA IV is highly expressed in the choriocapillaris, but not expressed in the retina. The R14W CA IV mutation (Rebello et al., Proc. Natl. Acad. Sci. USA 101:6617–6622, 2004) is located in the signal sequence. The subsequently discovered R219S CA IV mutation is located in the sequence of the mature protein. The biological effects of these mutations were studied in transfected COS cells. Although the signal sequence mutation had only a modest effect on total enzyme production (70% of normal), the R219S mutant protein was completely inactive. Both mutations induced upregulation of BiP, PERK, and CHOP, markers of ER stress. Both suppressed levels of production of other secretory proteins. Both dramatically induced apoptosis as measured by Annexin V binding and TUNEL staining. The markers of ER stress and the number of cells undergoing apoptosis were both greatly reduced by growth in 4-phenylbutyric acid or CA inhibitors, which appeared to act as nonspecific and specific chemical chaperones, respectively. We propose that autosomal dominant RP17 associated with both CA IV mutations is a protein folding disease produced by apoptosis in capillary endothelial cells that nourish the overlying retina. These results suggest that patients with RP17 may respond clinically to treatment with chemical chaperones. This research is supported by NIH grant DK40163 to W.S.S.

Targeting of Unfolded PhoA to the TAT Translocon of Escherichia coli

In Escherichia coli, the Tat system does not translocate Tat signal sequence fused PhoA (RR-PhoA), as it requires disulfide formation for folding. Here we show that such a RR-PhoA construct can be efficiently targeted to the Tat translocon, but the transport is not completed. RR-PhoA is detectable in a 580-kDa TatBC-containing complex, which is the first substrate-bound TatBC complex detected in a bacterial system so far. A second TatBC complex near 440 kDa comprises most of the TatB and TatC but is devoid of RR-PhoA. The targeting of PhoA to the Tat translocon depends on the twin-arginine motif and results in severe growth defects. This physiological effect is likely to be due to proton leakage at the cytoplasmic membrane. The results point to mechanistic incompatibilities of the Tat system with unfolded proteins such as RR-PhoA. There does not exist an intrinsic quality control at the TatBC complex itself, although correct folding is inevitable for Tat-dependent translocation.

RC-101, a Retrocyclin-1 Analogue with Enhanced Activity against Primary HIV Type 1 Isolates

Rhesus macaques express three theta-defensins (RTDs 1-3), cyclic octadecapeptides with antiviral and lectin-like properties. Corresponding theta-defensin genes exist and are expressed in humans, but a signal sequence mutation prevents the formation of mature theta-defensin peptides. Retrocyclin-1 is a theta-defensin peptide whose precursor is encoded by human theta-defensin pseudogenes. It can protect human peripheral blood lymphocytes from infection by R5 and X4 strains of HIV-1, and provides a molecular template for designing novel antiviral agents. In this study, we used JC53-BL reporter cells to assess the activity of retrocyclin-1 (RC-100) and several analogues against primary HIV-1 isolates, including R5 and R5X4 strains of subtypes A-D, CRF-01_AE, and recombinants. Each analogue differed from retrocyclin-1 by a single amino acid substitution: Gly --> Tyr in RC-106, RC-115, and RC-116, and Arg --> Lys in RC-101. Although the modification in RC-101 was chemically conservative, this peptide was significantly more potent than retrocyclin-1 across the panel of primary isolates. We performed surface plasmon resonance binding studies, using recombinant gp120 and CD4 produced in insect cells. Although RC-100 and RC-101 bound gp120 LAV/IIIB with a K(d) of 30-35 nM, they bound gp120 from CRF-01_AE strains (CM 235 and 93TH975.15) with K(d) values of 200-750 nM. Overall, our findings suggest that clade-related differences in gp120 glycosylation impact the ability of retrocyclin-1 to bind this viral glycoprotein, and modulate the peptides' ability to prevent HIV-1 infection. The performance of RC-101 suggests that additional "engineering" could further enhance the antiviral properties of theta-defensins.

Signal sequences influence membrane integration of the prion protein.

Biosynthesis of the prion protein at the endoplasmic reticulum generates multiple topological forms. The topology of an individual chain is determined first by the localization of the N terminus and then by potential integration of the transmembrane domain into the lipid bilayer. Here, we provide the first evidence that signal sequences affect the latter of these events by demonstrating that some but not other signal sequences and signal sequence mutations result in significant increases in the fraction of prion protein nascent chains that integrate into the lipid bilayer. Through analysis of the prolactin signal sequence, an especially poor integration effector, we find that the N terminal and hydrophobic regions of the signal sequence affect integration most significantly. Mutations in either region result in a considerable increase in the number of chains that integrate. The effect of the signal sequence cannot be attributed to timing of signal cleavage or the state of the ribosome membrane junction, parameters previously found to affect protein biogenesis. We also present evidence that signal sequences that are poor integration effectors can promote integration under experimental conditions that allow the nascent chain more time to integrate. These findings reveal a previously unappreciated relationship between signal sequences and transmembrane integration.

Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa.

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa.