FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1998476174> ?p ?o ?g. }

• W1998476174 endingPage "25208" @default.
• W1998476174 startingPage "25203" @default.
• W1998476174 abstract "The Glut1 glucose transporter is a glycoprotein whose membrane topology has been verified by a number of experimental observations, all of which are consistent with a 12-transmembrane helix model originally based on hydrophobicity analysis. We used Glut1 as a model multispanning membrane protein to test the Charge Difference Hypothesis (Hartmann, E., Rapoport, T. A., and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5786–5790), which asserts that the topology of a eucaryotic multispanning membrane protein is determined solely by the amino acid charge difference across the first transmembrane segment. The charge difference across the first transmembrane segment of Glut1 was progressively inverted in two independent series of mutants, one series in which only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number of negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first transmembrane segment does affect the topology of the protein, but that contrary to the hypothesis, it only dictates the orientation of the first transmembrane segment and the disposition of the amino terminus and the first linker domain. Charge inversion resulted in the formation of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane. The topology of downstream regions of Glut1 was unaffected by charge inversion across the first transmembrane segment, indicating that downstream sequences are important in determining the local topological disposition of the molecule. The Glut1 glucose transporter is a glycoprotein whose membrane topology has been verified by a number of experimental observations, all of which are consistent with a 12-transmembrane helix model originally based on hydrophobicity analysis. We used Glut1 as a model multispanning membrane protein to test the Charge Difference Hypothesis (Hartmann, E., Rapoport, T. A., and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5786–5790), which asserts that the topology of a eucaryotic multispanning membrane protein is determined solely by the amino acid charge difference across the first transmembrane segment. The charge difference across the first transmembrane segment of Glut1 was progressively inverted in two independent series of mutants, one series in which only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number of negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first transmembrane segment does affect the topology of the protein, but that contrary to the hypothesis, it only dictates the orientation of the first transmembrane segment and the disposition of the amino terminus and the first linker domain. Charge inversion resulted in the formation of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane. The topology of downstream regions of Glut1 was unaffected by charge inversion across the first transmembrane segment, indicating that downstream sequences are important in determining the local topological disposition of the molecule. uridin 5′-[α-35S]thiotriphosphate modified Barth's saline endoglycosidase H. The structural determinants for the membrane topology of bitopic and polytopic proteins expressed in Escherichia coli have been systematically and comprehensively explored by von Heijne (1von Heijne G. Mol. Microbiol. 1997; 24: 249-253Crossref PubMed Scopus (29) Google Scholar, 2von Heijne G. Prog. Biophys. Mol. Biol. 1996; 66: 113-139Crossref PubMed Scopus (89) Google Scholar, 3von Heijne G. Subcell. Biochem. 1994; 22: 1-19Crossref PubMed Scopus (39) Google Scholar). The topological assembly of membrane proteins is exceedingly complex and is influenced by a number of factors. However, the primary determinant of sec-dependent membrane protein topology in E. coli appears to be the distribution of positively charged amino acid side chains, with the positive charges apparently acting to prevent translocation of the amino- and carboxyl-terminal segments as well as the connecting loops of bitopic or polytopic proteins. This “positive-inside” rule is at least in part explainable by the negative-inside electrical charge across the E. colicytoplasmic membrane. In contrast, less is known about the topological determinants of eucaryotic membrane proteins, especially the more structurally complex multispanning proteins. Statistical analysis of a data base of inferred eucaryotic membrane protein topologies suggested that somewhat different rules may apply to eucaryotic multispanning proteins in comparison with their bacterial counterparts (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar, 5Sipos L. von Heijne G. Eur. J. Biochem. 1993; 213: 1333-1340Crossref PubMed Scopus (253) Google Scholar). Based on their analysis, Hartmann et al. (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar) proposed the Charge Difference Hypothesis, wherein the net amino acid electrical charge across the first transmembrane segment of a eucaryotic polytopic membrane protein determined its orientation: a net positive-cytoplasmic charge was proposed to dictate a cytoplasmic disposition for the amino terminus, and a net negative-cytoplasmic charge was proposed to dictate the opposite (extracytoplasmic) disposition. The remaining transmembrane segments were proposed to fold into the membrane in a passive, processive manner, such that downstream structural elements play little if any role in determining the final topology of the protein. Although this hypothesis is at least in part consistent with experimental observations made on an artificially constructed multispanning membrane protein (6Wessels H.P. Spiess M. Cell. 1988; 55: 61-70Abstract Full Text PDF PubMed Scopus (121) Google Scholar), it has not yet been systematically tested for a single, native, eucaryotic multispanning membrane protein. This is likely due to the fact that, until recently, the detailed topology of very few eucaryotic polytopic proteins had been experimentally established. Although the results of experiments conducted on the yeast Ste2p G protein-coupled receptor (7Harley C.A. Tipper D.J. J. Biol. Chem. 1996; 271: 24625-24633Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and artificial polytopic membrane proteins (8Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) are consistent with the charge difference hypothesis, studies with mutant bitopic proteins derived from the asialoglycoprotein receptor (9Beltzer J.P. Fiedler K. Fuhrer C. Geffen I. Handschin C. Wessels H.P. Spiess M. J. Biol. Chem. 1991; 266: 973-978Abstract Full Text PDF PubMed Google Scholar) or preprolactin (10Andrews D.W. Young J.C. Mirels L.F. Czarnota G.J. J. Biol. Chem. 1992; 267: 7761-7769Abstract Full Text PDF PubMed Google Scholar) suggest that the charge difference across a hydrophobic segment may not be the sole determinant of transmembrane orientation even for these topologically simpler proteins. We have recently (11Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar) used glycosylation-scanning mutagenesis to verify the 12-transmembrane helix topological model originally proposed for the Glut1 glucose transporter based on a hydrophobicity algorithm (12Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1140) Google Scholar). The 12-helix model for Glut1 is also consistent with a number of other experimental observations (13Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (32) Google Scholar). Glut1 is the structural prototype for a large family of membrane transporters, all of which are predicted to possess the same 12-helix topology (14Marger M.D. Saier Jr., M.J. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (750) Google Scholar). We have used Glut1 as a model to systematically investigate the charge difference hypothesis as it applies to a native multispanning membrane protein. We report herein that, consistent with the charge difference hypothesis, the amino acid charge difference across the first transmembrane segment is important in determining the disposition of the soluble amino-terminal domain, the first transmembrane segment, and the first exofacial loop. However, in contradiction to the charge difference hypothesis, downstream elements also play an important role in determining the final topology of this native multispanning membrane protein. Human Glut1 cDNA was previously subcloned into the oocyte expression vector pSP64T (15Krieg P.A. Melton D.A. Nucleic Acids Res. 1984; 14: 7057-7070Crossref Scopus (1081) Google Scholar). The aglyco-Glut1 construct (Asn45 mutated to Thr), into which the glycosylated exofacial domain of rat Glut4 was inserted independently into each of the putative hydrophilic soluble domains using engineered KpnI restriction sites, was previously subcloned into either the vector pSP64 or pSP64T and used as a probe for determining the protein topology (11Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). The amino acid sequence of the inserted epitope was as follows: GTNAPQKVIEQSYNATWLGRQGPGGPDSIPQGTLTTLWAGT. The epitope contains an N-linked glycosylation site, underlined above, and is flanked by Gly-Thr, the translated amino acid sequence corresponding to the KpnI restriction site. Two positively charged residues and two negatively charged residues shown in bold type were mutated to glycine residues in order to neutralize the charges within the marker sequence. Site-directed mutagenesis was performed using the CLONTECH Transformer Site Directed Mutagenesis kit (CLONTECH Laboratories, Palo Alto, CA). The sequences of the mutant transporters were verified using the Sequenase 2.0 kit (U. S. Biochemical Corp.). Mutant Glut1 mRNAs were synthesized from pSP64 or pSP64T cDNAs linearlized at a unique restriction site in the polylinker using the MEGAscript SP6 In Vitro Transcription kit (Ambion, Austin, TX). Manufacturer's conditions were modified by adding 4 mm GpppG and 0.625 mCi/ml [35S]UTPαS1(Amersham) and by reducing the GTP concentration to 1 mm. Ovarian lobes were resected from adult female Xenopus laevis (Nasco, Fort Atkinson, WI) anesthetized with 3-aminobenzonic acid ethyl ester (1 g/liter) in ice-cold water. The lobes were digested with 2 mg/ml Type I collagenase (Worthington) in calcium-free modified Barth's saline (MBS) at room temperature for 2 h. The oocytes were washed six times in MBS containing 5% bovine serum albumin and 50 μg/ml gentamicin and incubated at 4 °C in the same medium until injection. Stage V or IV oocytes were visually selected, injected with 50 nl of mRNA (1 μg/μl), and then incubated at 18 °C for 3 days in the same medium. Intracellular membranes were prepared and analyzed by Western blot analysis as described previously (16Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). 30 μg of intracellular membranes prepared from oocytes were solubilized in 1% SDS, adjusted to a final concentration of 0.25% SDS, 1.9% Nonidet P-40, 25 mm2-mercaptoethanol, 30 mm Tris-HCl, pH 8.0, and then digested with 0.25 unit of N-glycanase (Genzyme, Boston, MA) overnight at 37 °C. Groups of 15 oocytes were microinjected with the appropriate mutant Glut1 mRNA, incubated at 18 °C for 2 h, and then labeled with 2 mCi/ml Tran35S–label (ICN, Irvine, CA) in MBS at 18 °C for 1 h. The oocytes were extensively washed with MBS supplemented with 5 g/liter bovine serum albumin, 15 mm cold methionine, and 2 mm cysteine, and then intracellular membranes were prepared as described previously (16Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). Membranes were solubilized in 80 μl of 1% SDS and then diluted with 1 ml of 1% Triton X-100, 1% deoxycholate, 1% bovine serum albumin in phosphate-buffered saline, pH 7.4. Transporters were immunoprecipitated overnight at 4 °C with 1.25 μg of a monoclonal Glut1 antibody (17Allard W.J. Lienhard G.E. J. Biol. Chem. 1985; 260: 8668-8675Abstract Full Text PDF PubMed Google Scholar) (a kind gift of Dr. G. E. Lienhard, Darthmouth Medical School) precoupled to 40 μl of goat anti-mouse IgG affinity gel (Cappel, Organon Teknika, West Chester, PA). After washing the resin six times in phosphate-buffered saline, transporters were eluted with 50 μl of 1% SDS. Twenty μl of eluted transporters were digested with endoglycosidase H (Endo H) at 37 °C for 1 h with the addition of 0.5 μl of 3 m sodium acetate, pH 5.5, and 0.3 milliunit of enzyme (Boehringer Mannheim). Samples were then subjected to SDS-polyacrylamide gel electrophoresis. Polyacrylamide gels were destained and then radiochemically enhanced with 1 msalicylic acid. The charge difference hypothesis states that the membrane topology of a polytopic protein is determined solely by the amino acid charge difference across the first transmembrane segment (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar). The Glut1 glucose transporter conforms to the charge difference rule in that the net amino acid charge difference across the first transmembrane segment is −2.5 (exoplasmic with respect to cytoplasmic) when counting the 15 amino acid residues flanking the first charged residue predicted to lie outside of the membrane. This hypothesis makes at least two predictions that are readily testable by experimental manipulation: 1) inversion of the charge difference across the first transmembrane segment should result in a complete 180° inversion of the membrane topology; 2) the effect of the charge inversion should be independent of whether the number of positively charged residues or negatively charged residues is altered in the two hydrophilic flanking domains. We proceeded to test these two predictions by site-directed mutagenesis of cDNA encoding the human Glut1 glucose transporter, expression of the resulting mutant transporters in a Xenopus oocyte expression system, and determination of the membrane topology usingN-linked glycosylation as a marker for the disposition of specific sites on the protein. The charge difference mutations were initially introduced into either wild-type Glut1, which contains a naturally occurring site of N-linked glycosylation at Asn45 within the first exofacial loop, or a glycosylation-scanning mutant containing the glycosylated exofacial loop of Glut4 inserted into the amino terminus of Glut1 after Lys6 (see Fig. 1). These constructs will be referred to as the first exofacial loop reporter (L1) and the amino terminus reporter (NT), respectively. We demonstrated previously that the Glut4 exofacial loop marker provides the precise pattern of glycosylation predicted by our original 12-helix topological model when a comprehensive series of insertion mutants was expressed in Xenopus oocytes (11Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). The exofacial loop of Glut4 used as the glycosylation marker was mutated to remove its charged residues so that it acted as a neutral marker with respect to the charge difference across the transmembrane segment. Two series of charge difference mutants were introduced into each of the two Glut1 reporter molecules (see Table I). In the “P” series, the charge difference across the first transmembrane segment was progressively inverted from −2.5 to +2.5 for the first exofacial loop reporter or from −1.5 to +2.5 for the amino terminus reporter by progressively changing positively charged residues to neutral glycine residues in the amino-terminal domain and by changing uncharged residues in the first exofacial loop domain to lysine residues. In the “N” series, the charge difference was inverted by progressively changing negatively charged residues in the first exofacial loop domain to neutral asparagine residues and by changing uncharged residues in the amino-terminal domain to glutamate residues. The charge difference was determined according to the algorithm defined by Hartmann et al. (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar), in which only the 15 residues flanking the first charged residue outside of the membrane were included in the calculation. Comparison of the results obtained with the P versus the N series should thus allow an evaluation of the relative importance of flanking positive charges versus flanking negative charges in determining the orientation of a membrane helix.Table IMutations introduced into Glut1 for testing the effect of the charge difference across the first transmembrane helix on the membrane topologyMutant identificationAmino acid changesNet charge differenceLoop 1 reporterNone−2.5P1/L1K6G−1.5P2/L1K6G, K7G−0.5P3/L1K6G, K7G, A35K+0.5P4/L1K6G, K7G, A35K, V39K+1.5P5/L1K6G, K7G, A35K, V39K, I40K+2.5N1/L1E41N−1.5N2/L1E41N, E42N−0.5N3/L1E41N, E42N, G10E+0.5N4/L1E41N, E42N, G10E, T9E+1.5N5/L1E41N, E42N, G10E, T9E, L8E+2.5Amino terminus reporterNone−1.5P2/NTK7G−0.5P3/NTK7G, A35K+0.5P4/NTK7G, A35K, V39K+1.5P5/NTK7G, A35K, V39K, I40K+2.5N2/NTE41N−0.5N3/NTE41N, E42N+0.5N4/NTE41N, E42N, G10E+1.5N5/NTE41N, E42N, G10E, T9E+2.5N6/NTE41N, E42N, G10E, T9E, L8E+3.5Loop 2 reporterNone−2.5P5/L2K6G, K7G, A35K, V39K, I40K+2.5N5/L2E41N, E42N, G10E, T9E, L8E+2.5Loop 3 reporterNone−2.5P5/L3K6G, K7G, A35K, V39K, I40K+2.5N5/L3E41N, E42N, G10E, T9E, L8E+2.5Loop 6 reporterNone−2.5P5/L6K6G, K7G, A35K, V39K, I40K+2.5N5/L6E41N, E42N, G10E, T9E, L8E+2.5Loop 9 reporterNone−2.5P5/L9K6G, K7G, A35K, V39K, I40K+2.5N5/L9E41N, E42N, G10E, T9E, L8E+2.5Carboxyl terminus reporterNone−2.5P5/CTK6G, K7G, A35K, V39K, I40K+2.5N5/CTE41N, E42N, G10E, T9E, L8E+2.5Amino acid residues are indicated by the single-letter code. The first letter, the number, and the second letter represent the wild-type amino acid residue, the residue number, and the substituted residue, respectively. L1 is wild-type Glut1 transporter, which has aN-linked glycosylation site in the first exofacial domain (see Fig. 1). Mutants labeled NT and CT use the aglyco-Glut1 transporter with the first exofacial domain of Glut4 inserted into either the amino or carboxyl terminus. Mutants L2, L3, L6, and L9 have the epitope inserted into the correspondingly numbered linker domain. P1 to P5 represent mutants in which the charge was progressively inverted by altering only the number of positively charged residues in the flanking domains. N1 to N6 represent mutants in which the charge was progressively inverted by altering only the number of negatively charged residues in the flanking domains. The net charge differences across the first transmembrane segment were calculated for a segment of 15 amino acid residues beginning with the first charged residues on either side (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar). Open table in a new tab Amino acid residues are indicated by the single-letter code. The first letter, the number, and the second letter represent the wild-type amino acid residue, the residue number, and the substituted residue, respectively. L1 is wild-type Glut1 transporter, which has aN-linked glycosylation site in the first exofacial domain (see Fig. 1). Mutants labeled NT and CT use the aglyco-Glut1 transporter with the first exofacial domain of Glut4 inserted into either the amino or carboxyl terminus. Mutants L2, L3, L6, and L9 have the epitope inserted into the correspondingly numbered linker domain. P1 to P5 represent mutants in which the charge was progressively inverted by altering only the number of positively charged residues in the flanking domains. N1 to N6 represent mutants in which the charge was progressively inverted by altering only the number of negatively charged residues in the flanking domains. The net charge differences across the first transmembrane segment were calculated for a segment of 15 amino acid residues beginning with the first charged residues on either side (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar). The mutant Glut1 mRNAs were transcribed in vitro and then injected into Xenopus oocytes. Three days postinjection, total oocyte membranes were prepared and dissolved in a detergent buffer, and equal aliquots were either treated or not treated with N-glycanase and then subjected to immunoblot analysis using Glut1-specific antibody. The mobility of glycosylated Glut1 protein was increased by treatment with N-glycanase, which cleaves N-linked oligosaccharides from glycoproteins. Surprisingly, the data in Fig. 2demonstrate that inversion of the charge difference had no discernible effect on the glycosylation status of either reporter construct, suggesting that the mutations had no apparent effect on the cytosolic disposition of the amino terminus or the exoplasmic disposition of the first loop. However, increasing the charge difference from −2.5 or −1.5 to more positive values resulted in a decreased level of steady-state proteins, until a dramatic reduction in mutant Glut1 proteins was observed at a charge difference of +2.5 for the first loop or amino terminus reporter constructs. We suspected that inversion of the charge difference was indeed altering the topology of the mutant proteins and that the molecules with aberrant topologies had a decreased stability in oocytes relative to molecules with the normal topology. In fact, long-term exposure of the autoradiogram revealed the presence of small quantities of aberrantly glycosylated amino terminus for the P5 mutant in the amino terminus reporter (P5/NT), indicating that the charge inversion did indeed alter the topology of Glut1. In order to bypass problems caused by decreased stability of topologically aberrant molecules, we examined the topology of mutant proteins pulse-labeled for 1 h with Tran35S–label. The presence of the high mannose core oligosaccharides on the newly synthesized, pulse-labeled mutant transporters was detected by digestion of membrane protein with endoglycosidase H. As with the N-glycanase digestions described above, cleavage of the high mannose core oligosaccharide from the glycosylated protein increased the mobility of glycosylated transporter on SDS gels. Fig. 3demonstrates that mutant molecules with altered topologies were detected after pulse labeling. A small fraction of the first loop reporter was unglycosylated in the P4/L1 mutant with a charge difference of +1.5, and ∼75% of the P5/L1 mutant with a charge difference of +2.5 remained unglycosylated, indicating that a fraction of the molecules had first loop domains that were not translocated into the lumen of the endoplasmic reticulum membrane and were therefore not exposed to the glycosylation machinery. In comparison, ∼50% of the N5/L1 mutant with a charge difference of +2.5 remained unglycosylated. These data demonstrate that both positively and negatively charged amino acid residues can influence the topology of Glut1. Similarly, the amino terminus reporter showed aberrant glycosylation behavior as the charge difference was inverted by altering the number of positive or negative charges in the two flanking domains. In this case, however, the amino terminus of the reporter construct started to exhibit some core glycosylation at a charge difference of +1.5 (N4/NT) or +0.5 (P3/NT), and nearly complete glycosylation was observed at a charge difference of +2.5, indicating that the normally cytoplasmically disposed amino terminus had “flipped” into the exoplasm. Again, both positive and negative charges affected the topology, but alteration of the distribution of positive charges appeared to be somewhat more effective. The data for the two amino-terminal series constructs are presented quantitatively in Fig. 3 B. In order to determine whether the topological disruptions induced by the charge inversions represented a complete 180° inversion of Glut1 topology (see Fig. 5 D) or only local perturbations in topology (see Fig. 5, B and C), we examined the disposition of downstream loops in the N and P series mutants exhibiting the most extreme alterations in charge difference. The glycosylation marker was placed individually into loops 2, 3, 6, or 9, or into the carboxyl terminus of the Glut1 mutants (see Fig. 1 and Table I). The data in Fig. 4 demonstrate that neither the N5 nor P5 charge mutant exhibited any aberrant downstream topology. That is, the predicted downstream cytoplasmic domains (loops 2 and 6 and the carboxyl terminus) remained cytoplasmic and the predicted exoplasmic domains (loops 3 and 9) remained exoplasmic for both the P and N series mutants according to the observed patterns of glycosylation.Figure 4Endo H digestion of mutant transporters immunoprecipitated from solubilized intracellular membranes purified from metabolically labeled oocytes for the orientation of the downstream flanking domains. Oocytes were injected with mRNA from each of the mutants and then labeled with Tran35S–label as described under “Experimental Procedures.” Transporters were then immunoprecipitated and treated without (−) or with (+) Endo H. P1 to P5represent mutants in which the charge was progressively inverted by altering only the number of positively charged residues in the flanking domains. N1 to N5 represent mutants in which the charge was progressively inverted by altering only the number of negatively charged residues in the flanking domains. The net charge differences across the first transmembrane segment were calculated for a segment of 15 amino acid residues beginning with the first charged residues on either side (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The assembly of multispanning membrane proteins is at present poorly understood and is no doubt an extremely complex process. Although some progress has been made in understanding the assembly of eucaryotic multispanning membrane proteins (18Mothes W. Heinrich S.U. Graf R. Nilsson I. von H.G. Brunner J. Rapoport T.A. Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), the precise molecular events involved in the folding of proteins into the membrane have yet to be defined. Our data demonstrate that charge inversion across the first transmembrane segment only caused a local disruption in Glut1 topology, most likely corresponding to molecules in which either the first or second transmembrane domain failed to insert into the membrane (see Fig. 5, B and C). The structures shown in Fig. 5, B and C, are consistent with the following observations for the N5 or P5 mutants exhibiting a charge difference of +2.5. About 75% of the molecules (in the case of the P series mutant) possessed an amino terminus that had aberrantly “flipped” into the lumen of the endoplasmic reticulum and possessed a first loop domain that had aberrantly failed to translocate and therefore remained in the cytoplasm. The other ∼25% of the molecules also possessed an aberrantly exoplasmic amino terminus, but possessed a normally disposed exoplasmic first loop domain. Because the downstream topology of both types of molecules appeared to be completely normal, the first class of molecules must correspond to the structure shown in Fig. 5 C,and the second class of molecules must correspond to the structure shown in Fig. 5 B. These “frustrated” topologies, in which a transmembrane segment is “pulled out” of the membrane, have been observed previously for mutant bacterial membrane proteins whose topologies have been subjected to experimental manipulation by charge alterations (19Gafvelin G. von Heijne G. Cell. 1994; 77: 401-412Abstract Full Text PDF PubMed Scopus (142) Google Scholar). Despite our ignorance of the mechanisms involved, the positive-inside rule has proven to be very successful at predicting the topology of bacterial polytopic proteins (2von Heijne G. Prog. Biophys. Mol. Biol. 1996; 66: 113-139Crossref PubMed Scopus (89) Google Scholar). However, statistical analyses (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar) and experimental results (8Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 20Johansson M. Nilsson I. von Heijne G. Mol. Gen. Genet. 1993; 239: 251-256Crossref PubMed Scopus (57) Google Scholar) have suggested that somewhat different rules may apply for eucaryotic proteins, perhaps reflecting the lack of an electrical potential across the endoplasmic reticulum membrane. Hartmann et al. (4Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5786-5790Crossref PubMed Scopus (488) Google Scholar) suggested that the total amino acid charge difference across the first transmembrane segment, rather than only the distribution of lysines and arginines, is critical for determining the topology of eucaryotic polytopic proteins. Although the charge difference hypothesis was proposed nearly a decade ago, until now it has not been systematically tested on a native, eucaryotic multispanning membrane protein. Our results with Glut1, a protein with 12 presumably α-helical transmembrane segments, confirm some aspects of the charge difference hypothesis, but also indicate that the determinants of eucaryotic polytopic membrane topology are more complex than suggested by this hypothesis alone. Consistent with the charge difference hypothesis, our data demonstrate that the amino acid charge difference across the first transmembrane segment is important in determining the topology of Glut1, and that, unlike the situation in E. coli, the distribution of both positive and negative charges contributes to the overall effect, although positive charges were somewhat more influential with respect to topology than negative charges. However, contrary to the hypothesis, the effect of this charge difference is limited to determining the disposition of the amino-terminal domain, the first transmembrane segment, and the first linker domain. In fact, inverting the amino acid charge difference across the first transmembrane segment resulted in the production of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane, whereas the topology of the molecules from the second linker domain to the carboxyl terminus was normal. These observations indicate that downstream sequences are involved in determining the topology of Glut1. Interestingly, each transmembrane segment of Glut1 obeys the charge difference rule as well as the positive-inside rule, with the exception of segment 4, which has a net amino acid charge across it of 0. It is quite possible that, depending on the lengths of the flanking linker domains for a given protein, the charge difference across each transmembrane helix is important in determining the orientation of that helix and the disposition of its flanking domains. This is a hypothesis that is now readily testable for Glut1." @default.
• W1998476174 created "2016-06-24" @default.
• W1998476174 creator A5025464893 @default.
• W1998476174 creator A5054724495 @default.
• W1998476174 creator A5079562644 @default.
• W1998476174 date "1998-09-01" @default.
• W1998476174 modified "2023-09-30" @default.
• W1998476174 title "Testing the Charge Difference Hypothesis for the Assembly of a Eucaryotic Multispanning Membrane Protein" @default.
• W1998476174 cites W1506378995 @default.
• W1998476174 cites W1537087455 @default.
• W1998476174 cites W1565070195 @default.
• W1998476174 cites W1572001614 @default.
• W1998476174 cites W1640153047 @default.
• W1998476174 cites W1968451768 @default.
• W1998476174 cites W1974682887 @default.
• W1998476174 cites W1983464528 @default.
• W1998476174 cites W1985899857 @default.
• W1998476174 cites W1990081741 @default.
• W1998476174 cites W1996994910 @default.
• W1998476174 cites W2010928027 @default.
• W1998476174 cites W2031552181 @default.
• W1998476174 cites W2064864270 @default.
• W1998476174 cites W2071416115 @default.
• W1998476174 cites W2074783201 @default.
• W1998476174 cites W2087333595 @default.
• W1998476174 cites W2089841008 @default.
• W1998476174 cites W34285493 @default.
• W1998476174 cites W71052557 @default.
• W1998476174 doi "https://doi.org/10.1074/jbc.273.39.25203" @default.
• W1998476174 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9737982" @default.
• W1998476174 hasPublicationYear "1998" @default.
• W1998476174 type Work @default.
• W1998476174 sameAs 1998476174 @default.
• W1998476174 citedByCount "41" @default.
• W1998476174 countsByYear W19984761742012 @default.
• W1998476174 countsByYear W19984761742014 @default.
• W1998476174 countsByYear W19984761742015 @default.
• W1998476174 countsByYear W19984761742017 @default.
• W1998476174 countsByYear W19984761742018 @default.
• W1998476174 countsByYear W19984761742019 @default.
• W1998476174 countsByYear W19984761742020 @default.
• W1998476174 countsByYear W19984761742021 @default.
• W1998476174 crossrefType "journal-article" @default.
• W1998476174 hasAuthorship W1998476174A5025464893 @default.
• W1998476174 hasAuthorship W1998476174A5054724495 @default.
• W1998476174 hasAuthorship W1998476174A5079562644 @default.
• W1998476174 hasBestOaLocation W19984761741 @default.
• W1998476174 hasConcept C121332964 @default.
• W1998476174 hasConcept C12554922 @default.
• W1998476174 hasConcept C144647389 @default.
• W1998476174 hasConcept C185592680 @default.
• W1998476174 hasConcept C188082385 @default.
• W1998476174 hasConcept C41625074 @default.
• W1998476174 hasConcept C55493867 @default.
• W1998476174 hasConcept C62520636 @default.
• W1998476174 hasConcept C86803240 @default.
• W1998476174 hasConcept C95444343 @default.
• W1998476174 hasConceptScore W1998476174C121332964 @default.
• W1998476174 hasConceptScore W1998476174C12554922 @default.
• W1998476174 hasConceptScore W1998476174C144647389 @default.
• W1998476174 hasConceptScore W1998476174C185592680 @default.
• W1998476174 hasConceptScore W1998476174C188082385 @default.
• W1998476174 hasConceptScore W1998476174C41625074 @default.
• W1998476174 hasConceptScore W1998476174C55493867 @default.
• W1998476174 hasConceptScore W1998476174C62520636 @default.
• W1998476174 hasConceptScore W1998476174C86803240 @default.
• W1998476174 hasConceptScore W1998476174C95444343 @default.
• W1998476174 hasIssue "39" @default.
• W1998476174 hasLocation W19984761741 @default.
• W1998476174 hasOpenAccess W1998476174 @default.
• W1998476174 hasPrimaryLocation W19984761741 @default.
• W1998476174 hasRelatedWork W1993740563 @default.
• W1998476174 hasRelatedWork W2000148018 @default.
• W1998476174 hasRelatedWork W2032161274 @default.
• W1998476174 hasRelatedWork W2093666789 @default.
• W1998476174 hasRelatedWork W2095441493 @default.
• W1998476174 hasRelatedWork W2155593527 @default.
• W1998476174 hasRelatedWork W2239347838 @default.
• W1998476174 hasRelatedWork W2949641971 @default.
• W1998476174 hasRelatedWork W3110055093 @default.
• W1998476174 hasRelatedWork W3171224921 @default.
• W1998476174 hasVolume "273" @default.
• W1998476174 isParatext "false" @default.
• W1998476174 isRetracted "false" @default.
• W1998476174 magId "1998476174" @default.
• W1998476174 workType "article" @default.