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cDNA cloning of the translocon associated protein β-subunit in the chick cerebellum
Gene 201 (1997) 1–4
cDNA cloning of the translocon associated protein b-subunit in the chick cerebellum Ioannis Zarkadis a, Elena Vezyri a, Elias Kouvelas b, Demetrios Thanos c, Joseph Papamatheakis c, Aglaia Athanassiadou a,* a Department of Biology, Medical Faculty, University of Patras, 26110 Patras, Greece b Physiology, Medical Faculty, University of Patras, 26110 Patras, Greece c Institute of Molecular Biology and Biotechnology – FORTH and Department of Biology, University of Crete, Heraklion, Greece Received 10 October 1996; accepted 13 June 1997
Abstract The ‘translocon associated protein’ is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The b subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the b subunit from chick nervous tissue, namely cerebellum. Its deduced protein sequence is 91% homologous to both the canine and the human protein sequences, showing that the molecule is highly conserved. © 1997 Elsevier Science B.V. Keywords: Translocon; Endoplasmic reticulum; Protein transfer; Neural tissue; c-DNA cloning
1. Introduction The transport of many eucaryotic proteins across the membrane of the endoplasmic reticulum ( ER) is an integral part of their biosynthesis, and takes place after the newly synthesized polypeptides are ‘targeted’ to the ER membrane by signal sequence ( Walter and Cingappa, 1986; Rapoport, 1992). The transport takes place at specific sites, the translocons, which contain the ‘translocon-associated protein’ ( TRAP), a stoichiometric complex of four membrane proteins, two glycosylated (subunits a and b) and two non-glycosylated ones (subunits c and d) (Hartmann et al., 1993). TRAP a, previously called ‘signal sequence receptor’, is a major constituent of the ER membrane in various species and was shown to be directly involved in the in-vitro translocation of several secretory proteins (Hartmann et al., 1989). TRAP b [previously called * Corresponding author. Tel.: +30 61 997621; Fax: +30 61 991769; e-mail: [email protected] Abbreviations: ER, endoplasmic reticulum; TRAP, translocon-associated protein; SSRb, signal-sequence receptor, b-subunit; NGF, nerve growth factor. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 64 - 8
SSRb (Gorlich et al., 1990) or gp25H ( Wada et al., 1991)], c and d subunits are also transmembrane proteins. The canine a and b, the human b and the rat c and d subunits have been cloned by cDNA cloning (Bodescot and Brison, 1994). The primary structure of all four proteins has been determined, and a topological model for the tetrameric TRAP complex has been proposed: subunits a, b and d are single-spanning membrane proteins, whereas subunit c spans the membrane, probably, four times; each of the TRAPa and TRAPb carries two carbohydrate chains, and TRAPd contains a disulfide bridge. TRAPc has a charged domain between the second and third membrane-spanning regions, which may be responsible for the interaction with the other subunits of the complex (Hartmann et al., 1993). The function, however, of the TRAP complex is not, as yet, understood, and the processes in which it may play a role could involve retention of ER proteins ( Wada et al., 1991), translocation of a subclass of proteins, assembly of membrane protein complexes—possibly acting as a chaperone—or even an enzymatic activity required for the modification of nascent polypeptides (Hartmann et al., 1993).
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I. Zarkadis et al. / Gene 201 (1997) 1–4
2. Experimental and discussion We report here the cDNA cloning and sequence analysis of the b subunit of TRAP from the chick cerebellum. The clone was isolated from a lgt11 gene library, deriving from cerebellum RNA of 2-day-old chicks, after screening with a 1.13-kb P12–13 probe (Sajovic et al., 1987), which is related to the ‘NGF Inducible Large External’ adhesion molecule. The isolated cDNA was in fact the result of a fusion between two molecules, one of which contains a sequence highly homologous to P12–13 probe, and a second one, which is the TRAPb molecule. As a result of the fusion between the two different individual cDNAs, the TRAPb molecule was not complete within this cDNA, and part of the DNA sequence containing the signal sequence at the NH -terminus of the molecule was missing. In order 2 to obtain the missing DNA sequence, we performed a
polymerase chain reaction, using as template the total DNA of the lgt11 gene library and primers, the forward and reverse lgt11 primers, each in combination with a primer C∞, deriving from our TRAPb sequence (nucleotides 477–496). Only PCRs with primers lgt11 forward and C∞ gave a product with a length of 600 bp, which, after DNA sequencing, produced the missing NH2 part of the chick TRAPb sequence, namely the 5∞ untranslated region and the DNA sequence of the first two amino acids of the signal sequence. The TRAPb cDNA sequence is 812 nucleotides long ( Fig. 1), and it contains an open reading frame (ORF ), which extends over 549 nucleotides, with 84.5% homology to the DNA sequence of the canine TRAPb (Gorlich et al., 1990; Wada et al., 1991) and 82.5% homology to that of the human molecule (Bodescot and Brison, 1994). This high degree of homology, which may reflect an evolutionary conserved mode of function of the
Fig. 1. Nucleotide sequence of the TRAPb cDNA from chick cerebellum and deduced amino acid sequence. The signal sequence and the polyadenylation signal are underlined with single and double lines, respectively.
I. Zarkadis et al. / Gene 201 (1997) 1–4
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Fig. 2. Comparison of the amino acid sequences of the canine the human and the chick TRAPb subunits. Amino acid differences appear in bold letters, amino acids 1–17 represent signal sequences, transmembrane domains are underlined and the glycosilable Asn residues are indicated by asterisks.
TRAPb molecules, is not observed among TRAPa cDNAs from various eukaryotes, where, nevertheless, the overall topology and the distribution of charges are conserved (Hartmann and Prehn, 1994). The cDNA of the chick TRAPb has a signal sequence that shows a lower nucleotide homology of only 53% with the corresponding region of the canine gene and 55% with that of the human gene, whereas the homology between the canine and human signal sequences is 78.5%. The AUG in the chick cDNA appears in a position that conforms with the consensus sequence for translation, as determined by Kozak (1989), particularly concerning the presence of the purine A in position −3, the most highly conserved nucleotide in all eukaryotic mRNAs. At the protein level (Fig. 2), the 17-amino-acid chick signal sequence shares a 41% homology with both the respective canine and human regions. This homology refers to seven amino-acid positions conserved in all three protein sequences. However, the chick signal sequence contains a very hydrophobic central core, and its last residue, cysteine, is one with a small, uncharged side chain. The overall sequence around position +1 includes a cleavage site conforming to Von Heijne’s rule of cleavable signal sequences ( Von Heijne, 1983). The deduced protein sequence of the cleaved polypeptide consists of 166 amino acids, shows a 95.8% homology to the canine and 96.4% to the human TRAPb molecules and contains two glycosylation Asn sites and a transmembrane domain. In all three cDNAs, the canine, the human and the chick, transmembrane regions are identical, and their position in the polypeptide as well as the positions of the two Asn residues that are glycosilable are conserved (Fig. 2). All six amino-acid differences between the chick and the human deduced cleaved polypeptide and six of the seven amino-acid differences with respect to the canine one fall within the luminal region of the molecule, which is supposed to be responsible for the interaction with
the other subunits of the TRAP complex (Gorlich et al., 1990). The 5∞ and 3∞ flanking sequences of the chick gene have less than 40% homology with the respective regions of the other two genes and the polyadenylation signal at the 3∞ end of the molecule.
Acknowledgement We wish to thank G.M. Maniatis for comments on the manuscript, and I. Labropoulou and A. Kokkinos for secretarial and technical assistance, respectively.
References Bodescot, M., Brison, O., 1994. Cloning and sequence analysis of the beta subunit of the human translocon-associated protein. Biochim. Biophys. Acta 1217, 101–102. Gorlich, D., Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., Rapoport, T.A., 1990. The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by difunctional reagents. J. Cell Biol. 111, 2283–2294. Hartmann, E., Wiedmann, M., Rapoport, T.A., 1989. A membrane component of the endoplasmic reticulum that may be essential for protein translocation. EMBO J. 8, 2225–2229. Hartmann, E., Gorlich, D., Kotska, S., Otto, A., Kraft, R., Knespel, S., Burger, E., Rapoport, T.A., Prehn, S., 1993. A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur. J. Biochem. 214, 375–381. Hartmann, E., Prehn, S., 1994. The N-terminal region of the alphasubunit of the TRAP complex has a conserved cluster of negative charges. FEBS Lett. 349, 324–326. Kozak, M., 1989. The scanning model for thanslation: an update. J. Cell Biol. 108, 229–241. Rapoport, T.A., 1992. Transport of proteins across the endoplasmic reticulum membrane. Science 258, 931–936.
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I. Zarkadis et al. / Gene 201 (1997) 1–4
Sajovic, P., Ennulat, D., Shelanski, M., Greene, L.A., 1987. Isolation of NILE glycoprotein-related cDNA probes. J. Neurochem. 49, 756–763. Von Heijne, G., 1983. Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133, 17–21. Wada, I., Rindress, D., Cameron, P.H., Ou, W-J., Doherty, J.J., , IILouvard, D., Bell, A.W., Dignard, D., Thomas, D.Y., Bergeron,
J.J.M., 1991. SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem. 266, 19599–19610. Walter, P., Cingappa, V.R., 1986. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 2, 499–516.
cDNA cloning of the translocon associated protein b-subunit in the chick cerebellum Ioannis Zarkadis a, Elena Vezyri a, Elias Kouvelas b, Demetrios Thanos c, Joseph Papamatheakis c, Aglaia Athanassiadou a,* a Department of Biology, Medical Faculty, University of Patras, 26110 Patras, Greece b Physiology, Medical Faculty, University of Patras, 26110 Patras, Greece c Institute of Molecular Biology and Biotechnology – FORTH and Department of Biology, University of Crete, Heraklion, Greece Received 10 October 1996; accepted 13 June 1997
Abstract The ‘translocon associated protein’ is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The b subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the b subunit from chick nervous tissue, namely cerebellum. Its deduced protein sequence is 91% homologous to both the canine and the human protein sequences, showing that the molecule is highly conserved. © 1997 Elsevier Science B.V. Keywords: Translocon; Endoplasmic reticulum; Protein transfer; Neural tissue; c-DNA cloning
1. Introduction The transport of many eucaryotic proteins across the membrane of the endoplasmic reticulum ( ER) is an integral part of their biosynthesis, and takes place after the newly synthesized polypeptides are ‘targeted’ to the ER membrane by signal sequence ( Walter and Cingappa, 1986; Rapoport, 1992). The transport takes place at specific sites, the translocons, which contain the ‘translocon-associated protein’ ( TRAP), a stoichiometric complex of four membrane proteins, two glycosylated (subunits a and b) and two non-glycosylated ones (subunits c and d) (Hartmann et al., 1993). TRAP a, previously called ‘signal sequence receptor’, is a major constituent of the ER membrane in various species and was shown to be directly involved in the in-vitro translocation of several secretory proteins (Hartmann et al., 1989). TRAP b [previously called * Corresponding author. Tel.: +30 61 997621; Fax: +30 61 991769; e-mail: [email protected] Abbreviations: ER, endoplasmic reticulum; TRAP, translocon-associated protein; SSRb, signal-sequence receptor, b-subunit; NGF, nerve growth factor. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 64 - 8
SSRb (Gorlich et al., 1990) or gp25H ( Wada et al., 1991)], c and d subunits are also transmembrane proteins. The canine a and b, the human b and the rat c and d subunits have been cloned by cDNA cloning (Bodescot and Brison, 1994). The primary structure of all four proteins has been determined, and a topological model for the tetrameric TRAP complex has been proposed: subunits a, b and d are single-spanning membrane proteins, whereas subunit c spans the membrane, probably, four times; each of the TRAPa and TRAPb carries two carbohydrate chains, and TRAPd contains a disulfide bridge. TRAPc has a charged domain between the second and third membrane-spanning regions, which may be responsible for the interaction with the other subunits of the complex (Hartmann et al., 1993). The function, however, of the TRAP complex is not, as yet, understood, and the processes in which it may play a role could involve retention of ER proteins ( Wada et al., 1991), translocation of a subclass of proteins, assembly of membrane protein complexes—possibly acting as a chaperone—or even an enzymatic activity required for the modification of nascent polypeptides (Hartmann et al., 1993).
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I. Zarkadis et al. / Gene 201 (1997) 1–4
2. Experimental and discussion We report here the cDNA cloning and sequence analysis of the b subunit of TRAP from the chick cerebellum. The clone was isolated from a lgt11 gene library, deriving from cerebellum RNA of 2-day-old chicks, after screening with a 1.13-kb P12–13 probe (Sajovic et al., 1987), which is related to the ‘NGF Inducible Large External’ adhesion molecule. The isolated cDNA was in fact the result of a fusion between two molecules, one of which contains a sequence highly homologous to P12–13 probe, and a second one, which is the TRAPb molecule. As a result of the fusion between the two different individual cDNAs, the TRAPb molecule was not complete within this cDNA, and part of the DNA sequence containing the signal sequence at the NH -terminus of the molecule was missing. In order 2 to obtain the missing DNA sequence, we performed a
polymerase chain reaction, using as template the total DNA of the lgt11 gene library and primers, the forward and reverse lgt11 primers, each in combination with a primer C∞, deriving from our TRAPb sequence (nucleotides 477–496). Only PCRs with primers lgt11 forward and C∞ gave a product with a length of 600 bp, which, after DNA sequencing, produced the missing NH2 part of the chick TRAPb sequence, namely the 5∞ untranslated region and the DNA sequence of the first two amino acids of the signal sequence. The TRAPb cDNA sequence is 812 nucleotides long ( Fig. 1), and it contains an open reading frame (ORF ), which extends over 549 nucleotides, with 84.5% homology to the DNA sequence of the canine TRAPb (Gorlich et al., 1990; Wada et al., 1991) and 82.5% homology to that of the human molecule (Bodescot and Brison, 1994). This high degree of homology, which may reflect an evolutionary conserved mode of function of the
Fig. 1. Nucleotide sequence of the TRAPb cDNA from chick cerebellum and deduced amino acid sequence. The signal sequence and the polyadenylation signal are underlined with single and double lines, respectively.
I. Zarkadis et al. / Gene 201 (1997) 1–4
3
Fig. 2. Comparison of the amino acid sequences of the canine the human and the chick TRAPb subunits. Amino acid differences appear in bold letters, amino acids 1–17 represent signal sequences, transmembrane domains are underlined and the glycosilable Asn residues are indicated by asterisks.
TRAPb molecules, is not observed among TRAPa cDNAs from various eukaryotes, where, nevertheless, the overall topology and the distribution of charges are conserved (Hartmann and Prehn, 1994). The cDNA of the chick TRAPb has a signal sequence that shows a lower nucleotide homology of only 53% with the corresponding region of the canine gene and 55% with that of the human gene, whereas the homology between the canine and human signal sequences is 78.5%. The AUG in the chick cDNA appears in a position that conforms with the consensus sequence for translation, as determined by Kozak (1989), particularly concerning the presence of the purine A in position −3, the most highly conserved nucleotide in all eukaryotic mRNAs. At the protein level (Fig. 2), the 17-amino-acid chick signal sequence shares a 41% homology with both the respective canine and human regions. This homology refers to seven amino-acid positions conserved in all three protein sequences. However, the chick signal sequence contains a very hydrophobic central core, and its last residue, cysteine, is one with a small, uncharged side chain. The overall sequence around position +1 includes a cleavage site conforming to Von Heijne’s rule of cleavable signal sequences ( Von Heijne, 1983). The deduced protein sequence of the cleaved polypeptide consists of 166 amino acids, shows a 95.8% homology to the canine and 96.4% to the human TRAPb molecules and contains two glycosylation Asn sites and a transmembrane domain. In all three cDNAs, the canine, the human and the chick, transmembrane regions are identical, and their position in the polypeptide as well as the positions of the two Asn residues that are glycosilable are conserved (Fig. 2). All six amino-acid differences between the chick and the human deduced cleaved polypeptide and six of the seven amino-acid differences with respect to the canine one fall within the luminal region of the molecule, which is supposed to be responsible for the interaction with
the other subunits of the TRAP complex (Gorlich et al., 1990). The 5∞ and 3∞ flanking sequences of the chick gene have less than 40% homology with the respective regions of the other two genes and the polyadenylation signal at the 3∞ end of the molecule.
Acknowledgement We wish to thank G.M. Maniatis for comments on the manuscript, and I. Labropoulou and A. Kokkinos for secretarial and technical assistance, respectively.
References Bodescot, M., Brison, O., 1994. Cloning and sequence analysis of the beta subunit of the human translocon-associated protein. Biochim. Biophys. Acta 1217, 101–102. Gorlich, D., Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., Rapoport, T.A., 1990. The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by difunctional reagents. J. Cell Biol. 111, 2283–2294. Hartmann, E., Wiedmann, M., Rapoport, T.A., 1989. A membrane component of the endoplasmic reticulum that may be essential for protein translocation. EMBO J. 8, 2225–2229. Hartmann, E., Gorlich, D., Kotska, S., Otto, A., Kraft, R., Knespel, S., Burger, E., Rapoport, T.A., Prehn, S., 1993. A tetrameric complex of membrane proteins in the endoplasmic reticulum. Eur. J. Biochem. 214, 375–381. Hartmann, E., Prehn, S., 1994. The N-terminal region of the alphasubunit of the TRAP complex has a conserved cluster of negative charges. FEBS Lett. 349, 324–326. Kozak, M., 1989. The scanning model for thanslation: an update. J. Cell Biol. 108, 229–241. Rapoport, T.A., 1992. Transport of proteins across the endoplasmic reticulum membrane. Science 258, 931–936.
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I. Zarkadis et al. / Gene 201 (1997) 1–4
Sajovic, P., Ennulat, D., Shelanski, M., Greene, L.A., 1987. Isolation of NILE glycoprotein-related cDNA probes. J. Neurochem. 49, 756–763. Von Heijne, G., 1983. Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133, 17–21. Wada, I., Rindress, D., Cameron, P.H., Ou, W-J., Doherty, J.J., , IILouvard, D., Bell, A.W., Dignard, D., Thomas, D.Y., Bergeron,
J.J.M., 1991. SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem. 266, 19599–19610. Walter, P., Cingappa, V.R., 1986. Mechanism of protein translocation across the endoplasmic reticulum membrane. Annu. Rev. Cell Biol. 2, 499–516.