Combinatorial Control of Prion Protein Biogenesis by the Signal Sequence and Transmembrane Domain
2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês
10.1074/jbc.m101638200
ISSN1083-351X
AutoresSoo Jung Kim, Reza Rahbar, Ramanujan S. Hegde,
Tópico(s)RNA regulation and disease
ResumoThe prion protein (PrP) is synthesized in three topologic forms at the endoplasmic reticulum. secPrP is fully translocated into the endoplasmic reticulum lumen, whereasNtmPrP and CtmPrP are single-spanning membrane proteins of opposite orientation. Increased generation of CtmPrP in either transgenic mice or humans is associated with the development of neurodegenerative disease. To study the mechanisms by which PrP can achieve three topologic outcomes, we analyzed the translocation of proteins containing mutations introduced into either the N-terminal signal sequence or potential transmembrane domain (TMD) of PrP. Although mutations in either domain were found to affect PrP topogenesis, they did so in qualitatively different ways. In addition to its traditional role in mediating protein targeting, the signal was found to play a surprising role in determining orientation of the PrP N terminus. By contrast, the TMD was found to influence membrane integration. Analysis of various signal and TMD double mutants demonstrated that the topologic consequence of TMD action was directly dependent on the previous, signal-mediated step. Together, these results reveal that PrP topogenesis is controlled at two discrete steps during its translocation and provide a framework for understanding how these steps act coordinately to determine the final topology achieved by PrP. The prion protein (PrP) is synthesized in three topologic forms at the endoplasmic reticulum. secPrP is fully translocated into the endoplasmic reticulum lumen, whereasNtmPrP and CtmPrP are single-spanning membrane proteins of opposite orientation. Increased generation of CtmPrP in either transgenic mice or humans is associated with the development of neurodegenerative disease. To study the mechanisms by which PrP can achieve three topologic outcomes, we analyzed the translocation of proteins containing mutations introduced into either the N-terminal signal sequence or potential transmembrane domain (TMD) of PrP. Although mutations in either domain were found to affect PrP topogenesis, they did so in qualitatively different ways. In addition to its traditional role in mediating protein targeting, the signal was found to play a surprising role in determining orientation of the PrP N terminus. By contrast, the TMD was found to influence membrane integration. Analysis of various signal and TMD double mutants demonstrated that the topologic consequence of TMD action was directly dependent on the previous, signal-mediated step. Together, these results reveal that PrP topogenesis is controlled at two discrete steps during its translocation and provide a framework for understanding how these steps act coordinately to determine the final topology achieved by PrP. prion protein endoplasmic reticulum transmembrane domain N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine The prion protein (PrP)1is a 35-kDa brain glycoprotein involved in the transmission and/or pathogenesis of several neurodegenerative diseases, including scrapie, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease (1Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar, 2Johnson R.T. Gibbs Jr., C.J. N. Engl. J. Med. 1998; 339: 1994-2004Crossref PubMed Scopus (330) Google Scholar, 3Weissmann C. J. Biol. Chem. 1999; 274: 3-6Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Previous studies examining PrP biogenesis revealed that the normal protein is synthesized in three topologic forms at the endoplasmic reticulum (ER) (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 6Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The predominant form (termed secPrP) is fully translocated into the ER lumen, whereas the other two forms (termedNtmPrP and CtmPrP) are single-spanning membrane proteins. CtmPrP spans the membrane with its C terminus in the lumen, whereas NtmPrP is in the reverse orientation, with its N terminus in the lumen (see Fig. 1 Bfor a diagram). Mutations that result in increased generation of CtmPrP were shown to result in dose-dependent development of neurodegenerative disease (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). Additionally, a human disease causing mutation in PrP (A117V, resulting in Gerstmann-Sträussler-Scheinker disease (7Hsiao K.K. Cass C. Schellenbert G.D. Bird T. Devine-Gage E. Wisniewski H. Prusiner S.B. Neurology. 1991; 41: 681-684Crossref PubMed Scopus (139) Google Scholar, 8Tranchant C. Doh-ura K. Warter J.M. Steinmetz G. Chevalier Y. Hanauer A. Kitamoto T. Tateishi J. J. Neurol. Neurosurg. Psychiatry. 1992; 55: 185-187Crossref PubMed Scopus (50) Google Scholar, 9Mastrianni J.A. Curtis M.T. Oberholtzer J.C. DaCosta M.M. DeArmond S. Prusiner S.B. Garbern J.Y. Neurology. 1995; 45: 2042-2050Crossref PubMed Scopus (70) Google Scholar, 10Mallucci G.R. Campbell T.A. Dickinson A. Beck J. Holt M. Plant G. de Pauw K.W. Hakin R.N. Clarke C.E. Howell S. Davies-Jones G.A. Lawden M. Smith C.M. Ince P. Ironside J.W. Bridges L.R. Dean A. Weeks I. Collinge J. Brain. 1999; 122: 1823-1837Crossref PubMed Scopus (61) Google Scholar)) was shown to result in increased generation of CtmPrP in vitro, in mice (which also developed neurodegenerative disease), and in humans (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). Finally, recent studies have suggested that the ability to generate CtmPrP may also play a role in the neurodegeneration seen in transmissible forms of prion disease (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar). Thus, elevated levels of CtmPrP appear to be one mechanism by which PrP is able to mediate neurodegeneration. The role of CtmPrP in the pathogenesis of at least a subset of prion diseases highlights the importance of understanding the mechanisms by which PrP topology is determined and controlled. Generally, a protein's topology is thought to be unique and determined by "topogenic elements" encoded within the primary sequence (11Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1496-1500Crossref PubMed Scopus (908) Google Scholar, 12Wessels H.P. Spiess M. Cell. 1988; 55: 61-70Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 13Lipp J. Flint N. Haeuptle M.T. Dobberstein B. J. Cell Biol. 1989; 109: 2013-2022Crossref PubMed Scopus (56) Google Scholar). PrP contains three such topogenic sequences: an N-terminal signal sequence, generally used to target proteins to the ER (14Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (719) Google Scholar); a hydrophobic stretch of amino acids that can serve as a transmembrane domain (TMD); and a C-terminal sequence for glycolipid anchor addition (15Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (907) Google Scholar). However, not only do these elements fail to specify a homogeneous population of chains in a single topology, but the three topologic forms of PrP differ in two fundamental ways: localization of the N terminus (secPrP andNtmPrP have their N terminus in the ER lumen, whereasCtmPrP has it in the cytosol) and integration of the potential TMD into the lipid bilayer (secPrP is not integrated, whereas NtmPrP and CtmPrP are). The region(s) of PrP that encode the key determinants for each of the topologic forms have not been clearly elucidated. The N-terminal signal sequence is likely to be necessary for at least targeting PrP to the secretory pathway. This is supported by the observation that deletion or replacement of this domain results in PrP being made as a cytosolic protein (16Ma J. Lindquist S. Nat. Cell Biol. 1999; 1: 358-361Crossref PubMed Scopus (105) Google Scholar). 2S. J. Kim, R. Rahbar, and R. S. Hegde, unpublished data. Whether the signal plays any role in topogenesis beyond its targeting function has not been studied. By contrast, previous studies demonstrating that mutations within or immediately preceding the TMD can alter the topologic forms of PrP generated have implicated this domain in PrP topogenesis (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 6Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, the role of this domain in the generation of each of the topologic forms remains obscure. Finally, results from Stewart and Harris (6Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) demonstrating that various mutations in the C terminus of PrP do not significantly affect topology suggest that regions C-terminal to the TMD may not play a significant role in topogenesis. This is consistent with previous observations that replacement of the entire C-terminal domain of PrP with a protein domain from globin does not significantly affect the generation of any of the topologic forms (17DeFea K.A. Nakahara D.H. Calayag M.C. Yost C.S. Mirels L.F. Prusiner S.B. Lingappa V.R. J. Biol. Chem. 1994; 269: 16810-16820Abstract Full Text PDF PubMed Google Scholar). Thus, other than a poorly defined role for the TMD, relatively little is currently understood about either the domain(s) involved or their respective role(s) in directing PrP topogenesis. This study was undertaken to elucidate a conceptual framework for understanding PrP topogenesis that would both provide tools and serve as a starting point for future mechanistic studies. Rabbit reticulocyte lysate and dog pancreatic rough microsomes were prepared and used as described (Ref. 18Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 1: 85-91Abstract Full Text Full Text PDF Scopus (87) Google Scholar and references therein). Anti-PrP monoclonal antibody 3F4 was a gift from the laboratory of S. B. Prusiner. Restriction enzymes, other DNA-modifying enzymes, and SP6 RNA polymerase were from New England Biolabs Inc. RNase inhibitor was from Promega. Routine laboratory chemicals were of the highest quality available commercially from Sigma, Mallinckrodt Chemical Works, or ICN. Standard techniques were used in the creation of all plasmid constructs (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All constructs were made in the pSP64 vector (Promega). Signal sequence mutants were all derived from a modified wild-type Syrian hamster PrP construct that contained silent NheI and AatII sites introduced at codons 8 and 19, respectively. This plasmid was digested with the desired combination of BglII (immediately preceding the start codon), NheI, AatII, or PflMI (codon 26) and ligated to synthetic oligonucleotides encoding the desired mutations. Most of the TMD mutations were made by site-directed mutagenesis. The remaining TMD mutations were made by first introducing silent restriction sites (BstBI and NdeI) at codons 103 and 111 to facilitate replacement of selected regions surrounding the TMD with synthetic oligonucleotides encoding the desired mutations. Combinations of signal and TMD mutants were made by replacing the wild-type TMD region of the wild-type mature region (excised with either KpnI and XbaI orBsu36I and EcoRI) with the relevant mutant TMD. All constructs were verified by automated sequencing. In vitrotranscription with SP6 RNA polymerase, translation with rabbit reticulocyte lysate in the presence of [35S]methionine, and translocation into canine rough microsomal membranes have been described (Refs. 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar and 18Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 1: 85-91Abstract Full Text Full Text PDF Scopus (87) Google Scholar and references therein). Translations were carried out at 32 °C for 30 min. Proteolysis was with 0.5 mg/ml proteinase K for 60 min at 0 °C. Reactions were terminated with 5 mm phenylmethylsulfonyl fluoride and transferred into 10 volumes of 1% SDS and 0.1 m Tris (pH 8) preheated to 100 °C. Samples were either analyzed directly by SDS-polyacrylamide gel electrophoresis on 12% Tris/Tricine gels (20Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10479) Google Scholar) or immunoprecipitated with anti-PrP monoclonal antibody 3F4 prior to SDS-polyacrylamide gel electrophoresis as previously described (5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 18Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 1: 85-91Abstract Full Text Full Text PDF Scopus (87) Google Scholar). All of the translocation reactions shown were performed in the presence of a competitive peptide inhibitor of glycosylation (NH2-Asp-Tyr-Thr-COOH). Inhibition of glycosylation does not affect the ratios of topologic forms generated (5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). It does, however, simplify the analysis since each topologic form is then represented by a single band, rather than the heterogeneous banding pattern seen with variable glycosylation. Quantitative ratios of topologic forms were determined by analysis of digitized autoradiographs on either Kodak X-Omat or BioMax films. Band size (in pixels) multiplied by mean band density (subtracted for film background) was used to assign a value to each band, followed by calculation of the appropriate ratios. It should be noted that the relative effect of each mutant was consistently and reproducibly observed in multiple experiments (with an effect as little as 10% difference in the formation of a topologic form being readily detectable). However, the absolute amount of the topologic forms generated for any given construct (including the wild type) varied from experiment to experiment, depending on temperature, time of translation, and batch of in vitro translation extract and microsomal membranes used. This is consistent with the observation that PrP topogenesis is dependent on multiple protein factors in both the cytosol (21Lopez C.D. Yost C.S. Prusiner S.B. Myers R.M. Lingappa V.R. Science. 1990; 248: 226-229Crossref PubMed Scopus (114) Google Scholar) and ER membrane (18Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 1: 85-91Abstract Full Text Full Text PDF Scopus (87) Google Scholar). For this reason, the data in Figs. 2and 4 represent quantitative analysis of experiments in which all of the signal mutants (Fig. 2) or TMD mutants (Fig. 4) were analyzed simultaneously, in triplicate, to allow accurate and direct comparisons between the mutants. The raw data shown in Figs. 1 and 3 are from translation reactions performed at another time with different batches of reagents. Thus, although the relative differences between the mutants in these experiments and the quantitative analyses in Figs. 2and 4 are similar, the absolute amounts of each topologic form are somewhat different.Figure 4The relative amounts of the topologic forms generated by each of the TMD mutants shown in Fig. 3 A were quantitated, and the ratios between any two of the forms (CtmPrP/secPrP (A),CtmPrP/NtmPrP (B), andsecPrP/NtmPrP (C)) were plotted as described in the legend to Fig. 2. Ratios that were >20 or <0.05 are represented on the graph as either 20 or 0.05, respectively, and are indicated with asterisks. In addition, PrP mutants that have been shown in previous studies to result in neurodegenerative disease in transgenic mice (7Hsiao K.K. Cass C. Schellenbert G.D. Bird T. Devine-Gage E. Wisniewski H. Prusiner S.B. Neurology. 1991; 41: 681-684Crossref PubMed Scopus (139) Google Scholar, 8Tranchant C. Doh-ura K. Warter J.M. Steinmetz G. Chevalier Y. Hanauer A. Kitamoto T. Tateishi J. J. Neurol. Neurosurg. Psychiatry. 1992; 55: 185-187Crossref PubMed Scopus (50) Google Scholar) are indicated in boldface type. wt, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Mutational analysis of the PrP TMD.A, the sequence of PrP surrounding the TMD is shown, with mutated residues in boldface type. B, representative TMD mutants shown in A were analyzed for their effect on PrP translocation and topology as described in the legend to Fig. 1. PK, proteinase K.View Large Image Figure ViewerDownload Hi-res image Download (PPT) During the course of our ongoing studies of signal sequence function, we noticed that replacing the signal sequence of PrP with functional signal sequences from certain other secretory proteins (for example, prolactin and angiotensinogen) resulted in a change in the ratio of topologic forms.2 This observation raised the possibility that, in addition to facilitating targeting of nascent PrP to the ER, the signal sequence may play a role in PrP topogenesis. To explore this idea, we generated and analyzed the effect on topology of mutations introduced into the PrP signal sequence. Signal sequences generally contain three domains. The h-region, a feature that is common among all signal sequences (22von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar, 23von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (859) Google Scholar), forms the hydrophobic core of at least 6 amino acids. Preceding the h-region in many signals is the n-region, a polar and often charged domain that is at the amino terminus of a signal sequence. The c-region is composed of the amino acids immediately preceding the signal sequence cleavage site. The polar n-region of the PrP signal (residues 1–7) was chosen for mutagenesis for three primary reasons. First, the n-region is highly divergent among signals of different proteins, varying in both length (from 1 to 17 amino acids) and net charge (ranging from −2 to +4) (22von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1535) Google Scholar, 23von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (859) Google Scholar). Second, mutations disrupting the h-region often severely impair the obligate targeting function of a signal sequence (24Silhavy T.J. Benson S.A. Emr S.D. Microbiol. Rev. 1983; 47: 313-344Crossref PubMed Google Scholar). And third, the signal sequences from other proteins that affected PrP topology most notably differed from each other in the n-region, particularly charged residues. 3R. S. Hegde, unpublished data. Thus, we focused our mutagenesis primarily on those changes that alter the net charge of the n-region of PrP (Fig.1 A). A protease protection assay was used to assess the topology of the n-region signal sequence mutants synthesized in an in vitrotranslation and translocation system (5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar) (Fig. 1 B). In this assay, only PrP that is translocated across the microsomal membrane generates protease-protected species. The size of the fragment generated upon protease digestion indicates the topologic form from which it was derived (an ∼18-kDa C-terminal fragment fromCtmPrP and an ∼14-kDa N-terminal fragment fromNtmPrP), with protection of full-length PrP indicative ofsecPrP. As shown in Fig. 1 C, many of the signal sequence mutations resulted in a significant change in comparison with the wild type in the relative amounts of the protease-protected PrP fragments. The N1, N2, N6, N7, N7a, and N11 mutants generated, to varying degrees, increased amounts of the 18-kDa CtmPrP fragment upon protease digestion. By contrast, we consistently observed that the N4, N5, N10, and N12 mutants generated slightly lessCtmPrP than the wild type (see the quantitative analysis in Fig. 2 below). The N3 and N7a mutants, unlike the other mutants, showed a clear discrepancy in the amounts of the protease-protected fragments relative to the amount of synthesized PrP. In both cases, less than half of the synthesized PrP could be accounted for after protease digestion of the sample (Fig. 1 C, compare − PK and + PK lanes for these constructs). Presumably, these unaccounted chains are cytosolic and thus digested completely upon protease addition. This suggests that these two signal mutants, in addition to affecting the ratio of topologic forms generated, translocate less efficiently, resulting in some of the PrP remaining in the cytosol. Thus, although some mutations (e.g. N3) appear to reduce translocation efficiency, probably by affecting the targeting function of the signal sequence, other mutations (e.g. N2) appear to have a significant impact on topogenesis without an overall decrease in translocation efficiency. Together, these results suggest that, in addition to targeting, the signal may play a separate role in topogenesis. To gain additional insight into which aspects of PrP topogenesis were most influenced by mutations in the signal sequence, we quantified (see "Experimental Procedures") and plotted the relative ratios of the three topologic forms of PrP generated by the signal mutants that did not significantly affect translocation efficiency (e.g. all mutants except N3 and N7a). As shown in Fig.2, theCtmPrP/secPrP ratio varied dramatically between the different mutants, spanning an order of magnitude from ∼0.17 (e.g. N12) to ∼1.7 (e.g. N6) (Fig.2 A). The CtmPrP/NtmPrP ratio similarly ranged from ∼0.28 to ∼2.1 with these same mutants (Fig.2 B). By marked contrast, thesecPrP/NtmPrP ratio was surprisingly invariant, being between ∼1.1 and 1.8 regardless of the mutation analyzed (Fig.2 C). These results suggest that the primary effect on topogenesis of signal sequence mutations is to increase or decrease the amount ofCtmPrP relative to both NtmPrP andsecPrP. We have not observed a significant change in the ratio of NtmPrP to secPrP upon manipulation of the signal sequence with either mutations (Figs. 1 and 2) or replacement with other signal sequences.2 Consequently, the amounts of NtmPrP and secPrP, relative toCtmPrP, appear to either both increase (e.g. with N4) or both decrease (e.g.N2) with mutations in the signal sequence. Two additional observations from the analysis of these mutants are noteworthy. First, introduction of charged residues into the n-region of the signal sequence generally alters the topogenesis of PrP. This does not appear to be due to disruption of an existent sequence motif, but rather an effect of the introduced charge. Thus, replacement of residues 4 and 5 with Asn or Gln (N13 and N14, respectively) does not alter topology, whereas Asp, Glu, Arg, or Lys (N2, N11, N5, and N12, respectively) significantly alters topology. Second, acidic residues in the n-region often result in increased CtmPrP, and basic residues generally favor decreased CtmPrP. However, this was not universally true. Introduction of Asp at position 4 (N7) resulted in a significant increase in CtmPrP, whereas the same change at position 5 (N8) had a minimal effect on topology. Additionally, basic residues at positions 6 and 7 (N6) increasedCtmPrP significantly. We next performed a similar analysis of mutations either within or immediately preceding the hydrophobic stretch of amino acids (residues 113–135) thought to compose the membrane-spanning domain in bothNtmPrP and CtmPrP (5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 6Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Previous studies have shown that mutations in this region of PrP can not only affect topology, but can also result in neurodegenerative disease (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar). However, the number of mutants analyzed was not sufficient to allow general conclusions to be drawn about the role of this domain in determining specific aspects of PrP topology. Over the past several years, numerous mutations have been made in this region for a variety of reasons, many unrelated to studies of PrP topogenesis. We took advantage of these existent mutants (Fig.3 A) to perform a careful analysis of their effects on PrP topogenesis. Just as with the signal sequence mutants, we reasoned that a systematic pattern may emerge that could provide insight into the role of the TMD in determining PrP topology. The autoradiographs of representative TMD mutants show that, consistent with previous observations (4Hegde R.S. Tremblay P. Groth D. DeArmond S.J. Prusiner S.B. Lingappa V.R. Nature. 1999; 402: 822-826Crossref PubMed Scopus (269) Google Scholar, 5Hegde R.S. Mastrianni J.A. Scott M.R. DeFea K.A. Tremblay P. Torchia M. DeArmond S.J. Prusiner S.B. Lingappa V.R. Science. 1998; 279: 827-834Crossref PubMed Scopus (616) Google Scholar, 6Stewart R.S. Harris D.A. J. Biol. Chem. 2001; 276: 2212-2220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), mutations within or preceding the TMD can significantly affect PrP topology (Fig. 3 B). However, it was also apparent that mutations in the TMD affect PrP topology in a qualitatively different manner than the signal mutants. First, unlike the signal mutants, several TMD mutants make essentially all of the translocated PrP in the secPrP form (e.g. G123P, ΔSTE, and, to a lesser extent, A120G). And second, in contrast to some of the signal mutants (e.g. N3 and N7a), none of the 17 TMD mutants analyzed generated exclusivelyCtmPrP or affected translocation efficiency. To consolidate these observations, we quantitated and graphed the relative ratios of the topologic forms generated by each of the TMD mutants (Fig. 4). We found that theCtmPrP/secPrP ratio varied from 20 (Fig. 4 C). By comparison, theCtmPrP/NtmPrP ratio was less variant, ranging from ∼0.4 to ∼2.3, with the majority of mutants showing a ratio of close to 1 (Fig. 4 B). Therefore, it appears that the primary effect of mutations in the TMD region is to alter the amounts of the topologic forms that are membrane-integrated (NtmPrP andCtmPrP) relative to the fully translocatedsecPrP. For example, some mutations, such as G123P and ΔSTE, make almost exclusively the secPrP form. Others, such as KH-II, AV3, and A120L, make relatively littlesecPrP in favor of increased NtmPrP andCtmPrP. These results suggest that the major role for the TMD in PrP topogenesis is to specify the percent of PrP chains that integrate into the lipid bilayer. The above analyses identified two domains within PrP that each contribute to the determination of topology. Although both of these domains affect topology, the quantitative analyses (Figs. 2 and 4) suggest that they affect it in qualitatively different ways. The general distinction is that the signal sequence increases or decreases CtmPrP relative to the other two topologic forms, whereas the TMD increases or decreases secPrP relative to the other two topologic forms. Given that the signal and TMD affect PrP topogenesis in different ways, it was conceivable that the effect of one domain is dominant to the other. Alternatively, the two domains may both contribute to PrP topogenesis by acting either sequentially or in concert. To determine the manner in which the signal and TMD mutants interact with each other, we generated and analyzed an array of PrP double mutants that contain various combinations of the signal sequence and TMD mutations (Fig. 5). We analyzed constructs that combined various signal sequence mutations with either of two TMD mutants: PrP(A120L) and PrP(ΔSTE) (Fig. 5,A and B). In addition, we also examined constructs in which various TMD mutatio
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