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Cloning and sequence analysis of the β subunit of the human translocon-associated protein
Biochimica et Biophysica Acta, 1217 (1994) 101-102
101
© 1994 Elsevier Science B.V. All rights reserved 0167-4781/94/$07.00
BBAEXP 90600
Short Sequence-Paper
Cloning and sequence analysis of the/3 subunit of the human translocon-associated protein Myriam Bodescot * and Olivier Brison Laboratoire d'Oncologie Mol~culaire, CNRS URA 1158, lnstitut Gustave Roussy, 94805 Villejuif (France) (Received 7 September 1993)
Key words: Translocon-associated protein; Gene cloning; Gene sequence; (Human)
A cDNA corresponding to the/3 subunit of the human translocon-associated protein was cloned and sequenced. The polypeptide is 183 amino acids long and 96% homologous to its canine counterpart. Both polypeptides contain a cleavable signal sequence, an NHz-terminal domain extruding in the endoplasmic reticulum lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm.
Synthesis of many eukaryotic proteins involves translocation of the nascent polypeptide across the endoplasmic reticulum (ER) membrane, a process triggered by the signal sequence [1]. When it emerges from the ribosome, the signal sequence binds to the signal recognition particle (SRP). The resulting complex is targeted to the E R membrane through the SRP receptor. After release of the signal sequence from the SRP, the nascent polypeptide interacts with a number of E R membrane proteins, including the translocon-associated protein (TRAP), previously-named the signal sequence receptor [2-8]. Even though some of these proteins are directly involved in the transport of the nascent polypeptide through the membrane, the role of the T R A P remains to be elucidated [3,5]. After crossing the E R membrane, the signal sequence is cleaved and the nascent polypeptide extrudes into the E R lumen. The T R A P is a stoichiometric complex of four subunits, a, /3, 7 and 6 [1]. The canine a and /3 subunits and the rat 3' and 15 subunits have been cloned [6-8]. We report the nucleotide sequence of a cDNA corresponding to the human /3 subunit. In the process of characterizing c - m y c cDNA clones isolated from a human colon carcinoma cell line (SW613-S) cDNA library, we found a clone containing a c - m y c cDNA located next to a cDNA homologous to the canine T R A P /3 subunit cDNA. The human and * Corresponding author. Fax: + 33 1 45596439. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X74104.
SSDI 01 6 7 - 4 7 8 1 ( 9 3 ) E 0 2 4 7 - L
canine cDNAs are 1089 and 1036 bp long, respectively (Fig. 1). The human and canine open reading frames are 91% (502/549) homologous at the nucleotide level and 96% (175/183) homologous at the amino acid level, with five conservative and three non-conservative changes (Fig. 2). The 5' and 3' human untranslated regions are respectively 64% (27/42) and 76% (339/445) homologous to the corresponding regions of the canine cDNA, with several additional or missing nucleotides. The nucleotide sequence around the putative initiator A T G is in agreement with Kozak's consensus sequence [9]. An in frame A T G is located nine nucleotides upstream from the putative initiator A T G in the human cDNA. This A T G is not present in the canine cDNA and its surrounding sequence does not conform to Kozak's consensus sequence. A direct repeat of a 22-bp sequence is located 17 bp downstream from the stop codon in the human cDNA, whereas the canine cDNA contains a single copy of the corresponding sequence. The human and canine cDNAs contain a polyadenylation signal at the same location, followed by a poly(A) tail. Analysis of the amino acid sequence and in vitro transcription and translation experiments have demonstrated that the canine polypeptide contains a cleavable signal sequence, an NH2-terminal domain extruding in the E R lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm [7]. The canine signal sequence is 17 amino acids long [7]. The human polypeptide shows three conservative and two non-conservative changes in the corresponding region. Both the human and canine signal sequences have the typical characteristics of a cleavable signal
102 GGCTCTCTTCCTGTCTTTGTGGCTCCGGAAAGGCGTTTGGGAIGCCAACG
50
ATG AGG CTG CIG TCA TTT GTG GTG fIG GCT CTA TTT GCT GIC Met Arg Leu Leu Ser Phe Val Val Leu Ala Leu Phe Ala Val
92
ACT CAA GCA GAG GAA GGA GCC AGG CTT TTG GCT TCC AAA TCA Thr Gln Ala Glu Glu Gly Ala Arg Leu Leu Ala Ser Lys Ser
134
CTG CTG AAC AGA TAC GCC GTG GAG GGA CGA GAC CTG ACC TTG Leu Leu Ash Arg Tyr Ala Val G1u Gly Arg Asp Leu Thr Leu
176
CAG TAC AAC ATC TAC AAT GTT GGC TCA AGT GCT GCA TTA GAC Gin Tyr Asn Ile Tyr Asn Val Gly Ser Ser Ala Ala Leu Asp
218
GTG GAA CTA TCT GAT GAT TCC TTC CCT CCA GAA GAC ITT GGC Val Glu Leu Ser Asp Asp Ser Phe Pro Pro Glu Asp Phe Gly
260
ATT GIG TCT GGA ATG CIC AAT GTC AAA TGG GAC CGG ATT GCC Ile Val Ser Gly Met Leu Ash Val Lys Trp Asp Arg lle Ala
302
CCT GCT AGC AAT GTC TCC CAC ACT GTG GIC CIG CGC CCT CTC Pro Ala Ser Ash Val Ser His lhr Val Val Leu Arg Pro keu
344
AAG GCT GGT TAT ITC AAC TTC ACC TCG GCA ACA ATT ACT TAC Lys Ala Gly Tyr Phe Asn Phe lhr Ser Ala Thr lle Thr Tyr
386
CTG GCC CAG GAG GAI GGG CCC GTT GTG ATT GGC TCT ACC AGT Leu Ala Gln Glu Asp Gly Pro Val Val lle Gly Ser Thr Ser
428
GCA CCT GGA CAG GGA GGA ATC CTG GCT CAG CGG GAG TIT GAC Ala Pro Gly Gin Gly Gly lle Leu Ala Gin Arg Glu Phe Asp
470
AGG CGA TTC TCC CCI CA1 ITT CTG GAC TGG GCA GCC TIT GGG Arg Arg Phe Ser Pro His Phe Leu Asp Trp Ala Ala Phe Gly
512
GTC ATG ACC CTT CCC TCC ATC GGC ATC CCC CTG CTA TTG TGG Val Met Thr Leu Pro Ser lle Gly lle Pro Leu Leu Leu Trp
554
TAC TCC AGC AAG AGG AAA TAT GAC ACT CCC AkJ~ACG AAG AAG Tyr Ser Ser Lys Arg Lys Tyr Asp Thr Pro Lys Thr Lys Lys
596
AAC TGATTGGGGCTTCCACAGCCCTCCTCTCCCAAGAAATCCAGGCTCCTCTCC Asn > >
650
CAAG#J~ATCCAGGTGCTTTCCAGACTCCA$b~GGGTATCTTAk.ATGCk.~TCTCTTC 705 TCTCTTAGCCCTTGGCCACTTTCTCCTGGATCCTGCCCTGCTCTCAGCCATAGTG 160 AAGGACCAGCCCTAGGAGTCTGCGAGAGCCTCCTTGGTTCCATCGTGAAGCCATA 815 AACAGGAATGCCTTTGGCAATAGCCTTGAGCCTAGAGGGCCCTCTGATGCCCCAC 870 TGAGGTGCTGTTGGTTTATTGCTGGCAACGTGAATTCTCTCAGGGGTCTAGGAGG 925 GGCATTTTGGAGACTGCCTGACACCACCCCTATCCCCTGCCTCCCCCTCTCAGAA 980 GAGGGTGGAAGATGAAJ~TGAAAGCTATGGGACTCTTGGAGGATACCCAGTGTCTA 1 0 3 5 TTCTGGGTTAGAGAAGTGCTTACTAAGGGGTTTTCTAATAAAAACAAATGCCAC 1089
Fig. 1. Nucleotide sequence of the human TRAP/3 subunit cDNA and deduced amino acid sequence. The first nucleotide of each
22-bp repeat is indicated by an arrow. The polyadenylation signal is underlined. Accession number X74104.
sequence, including a stretch of hydrophobic amino acid residues and a cleavage site conforming to Von Heijne's rule [10]. The amino acid sequences of the human and canine transmembrane domains are identi]0 20 30 40 50 60 70 MRLLSFVVLALFAVTQAEEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSFPPEDFG MRLLASVLLALFAVSHAEEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSFPPEDFG +
--
+
+
+
-
+_
.
.
80 90 100 110 120 130 140 [VSGMLNVKWDRIAPASNVSHTVVLRPLKAGYFNFTSATITYLAQEDGPVVIGSTSAPGQGGI LAQREFD ..................................................................... IVSGMLNVKWDRIAPASNVSHTVVLRPLKAGYFNFTSATVTYLAQEDGPVVIGFTSAPGQGGI LAQREFD +
+
*
+
+
*
__
+_
_
150 160 170 180 RRFSPHFLDWAAFGVMTLPSIGIPLLLWYSSKRKYDTPKTKKN ....................................... RRFSPHFLDWAAFGVMTLPSIGI PLLkWYSSKRKYDTPKSKKN ++
+++
-
+
++
Fig. 2. Comparison of the amino acid sequences of the human (top) and canine (bottom) T R A P / 3 subunits. Identical amino acid residues are indicated by a double dash, conservative changes by a single dash and non-conservative changes by the absence of a dash. The signal sequence and the transmembrane domain are underlined. The glycosylated gln residues are indicated by asterisks. The positively and negatively charged amino acid residues are indicated by + and - .
cal. Secondary structure predictions indicated that the canine polypeptide contains /3 sheets adjacent to the transmembrane domain in the luminal region, which could be responsible for the interaction with the other subunits [7]. The luminal regions of the human and canine polypeptides differ by two amino acid residues, with one conservative and one non-conservative change. The canine polypeptide contains two glycosylated gln residues in the luminal region [7], which are conserved in the human polypeptide. The human and canine polypeptides show no difference with respect to their positively and negatively charged amino acid residues. The 3'-terminal nucleotide sequence of the human TRAP/3 subunit c D N A is 96% (334/348 and 422/438) homologous to the partial nucleotide sequences of two anonymous cDNAs randomly cloned from human fetal and infant brain c D N A libraries (accession numbers M78597 and T03363, respectively) [11,12]. The sense strand of the human TRAP/3 subunit c D N A sequence is homologous to the antisense strands of the anonymous sequences. This probably indicates that the anonymous cDNAs were sequenced from their poly(A) tails [12] and that the resulting sequences were recorded as such. The nucleotide sequence differences are likely to be due to sequencing errors, since only one strand of each anonymous c D N A was sequenced [12] and the number of differences increases as the distance from the poly(A) tail increases. References 1 Rapoport, T.A. (1992) Science 258, 931-936. 2 Wiedmann, M., Kurzchalia, T.V., Hartmann, E. and Rapoport, T.A. (1987) Nature 328, 830-833. 3 Hartmann, E., Wiedmann, M. and Rapoport, T.A. (1989) EMBO J. 8, 2225-2229. 4 Vogel, F., Hartmann, E., G6rlich, D. and Rapoport, T.A. (1990) Eur. J. Cell Biol. 53, 197-202. 5 Migliaccio, G., Nicchitta, C.V. and Blobel, G. (1992) J. Cell Biol. 117, 15-25. 6 Prehn, S., Herz, J., Hartmann, E., Kurzchalia, T.V., Frank, R., Roemiscb, K., Dobberstein, B. and Rapoport, T.A. (1990) Eur. J. Biochem. 188, 439-445. 7 G6rlich, D., Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B. and Rapoport, T.A. (1990)J. Cell Biol. 111, 2283-2294. 8 Hartmann, E., G6rlich, D., Kostka, S., Otto, A.~ Kraft, R., Knespel, S., Biirger, E., Rapoport, T.A. and Prehn, S. (1993) Eur. J. Biochem. 214, 375-381. 9 Kozak, M. (1989) J. Cell Biol. 108, 229-241. 10 Von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21. 11 Adams, M.D., Dubnick, M., Kerlavage, A.R., Moreno, R., Kelley, J.M., Utterbach, T.R., Nagle, J.W., Fields, C. and Venter, J.C. (1992) Nature 355, 632-634. 12 Khan, A.S., Wilcox, A.S., Polymeropoulos, M.H., Hopkins, J.A., Stevens, T.J., Robinson, M., Orpana, A.K. and Sikela, J.M. (1992) Nature Genet. 2, 180-185.
101
© 1994 Elsevier Science B.V. All rights reserved 0167-4781/94/$07.00
BBAEXP 90600
Short Sequence-Paper
Cloning and sequence analysis of the/3 subunit of the human translocon-associated protein Myriam Bodescot * and Olivier Brison Laboratoire d'Oncologie Mol~culaire, CNRS URA 1158, lnstitut Gustave Roussy, 94805 Villejuif (France) (Received 7 September 1993)
Key words: Translocon-associated protein; Gene cloning; Gene sequence; (Human)
A cDNA corresponding to the/3 subunit of the human translocon-associated protein was cloned and sequenced. The polypeptide is 183 amino acids long and 96% homologous to its canine counterpart. Both polypeptides contain a cleavable signal sequence, an NHz-terminal domain extruding in the endoplasmic reticulum lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm.
Synthesis of many eukaryotic proteins involves translocation of the nascent polypeptide across the endoplasmic reticulum (ER) membrane, a process triggered by the signal sequence [1]. When it emerges from the ribosome, the signal sequence binds to the signal recognition particle (SRP). The resulting complex is targeted to the E R membrane through the SRP receptor. After release of the signal sequence from the SRP, the nascent polypeptide interacts with a number of E R membrane proteins, including the translocon-associated protein (TRAP), previously-named the signal sequence receptor [2-8]. Even though some of these proteins are directly involved in the transport of the nascent polypeptide through the membrane, the role of the T R A P remains to be elucidated [3,5]. After crossing the E R membrane, the signal sequence is cleaved and the nascent polypeptide extrudes into the E R lumen. The T R A P is a stoichiometric complex of four subunits, a, /3, 7 and 6 [1]. The canine a and /3 subunits and the rat 3' and 15 subunits have been cloned [6-8]. We report the nucleotide sequence of a cDNA corresponding to the human /3 subunit. In the process of characterizing c - m y c cDNA clones isolated from a human colon carcinoma cell line (SW613-S) cDNA library, we found a clone containing a c - m y c cDNA located next to a cDNA homologous to the canine T R A P /3 subunit cDNA. The human and * Corresponding author. Fax: + 33 1 45596439. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X74104.
SSDI 01 6 7 - 4 7 8 1 ( 9 3 ) E 0 2 4 7 - L
canine cDNAs are 1089 and 1036 bp long, respectively (Fig. 1). The human and canine open reading frames are 91% (502/549) homologous at the nucleotide level and 96% (175/183) homologous at the amino acid level, with five conservative and three non-conservative changes (Fig. 2). The 5' and 3' human untranslated regions are respectively 64% (27/42) and 76% (339/445) homologous to the corresponding regions of the canine cDNA, with several additional or missing nucleotides. The nucleotide sequence around the putative initiator A T G is in agreement with Kozak's consensus sequence [9]. An in frame A T G is located nine nucleotides upstream from the putative initiator A T G in the human cDNA. This A T G is not present in the canine cDNA and its surrounding sequence does not conform to Kozak's consensus sequence. A direct repeat of a 22-bp sequence is located 17 bp downstream from the stop codon in the human cDNA, whereas the canine cDNA contains a single copy of the corresponding sequence. The human and canine cDNAs contain a polyadenylation signal at the same location, followed by a poly(A) tail. Analysis of the amino acid sequence and in vitro transcription and translation experiments have demonstrated that the canine polypeptide contains a cleavable signal sequence, an NH2-terminal domain extruding in the E R lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm [7]. The canine signal sequence is 17 amino acids long [7]. The human polypeptide shows three conservative and two non-conservative changes in the corresponding region. Both the human and canine signal sequences have the typical characteristics of a cleavable signal
102 GGCTCTCTTCCTGTCTTTGTGGCTCCGGAAAGGCGTTTGGGAIGCCAACG
50
ATG AGG CTG CIG TCA TTT GTG GTG fIG GCT CTA TTT GCT GIC Met Arg Leu Leu Ser Phe Val Val Leu Ala Leu Phe Ala Val
92
ACT CAA GCA GAG GAA GGA GCC AGG CTT TTG GCT TCC AAA TCA Thr Gln Ala Glu Glu Gly Ala Arg Leu Leu Ala Ser Lys Ser
134
CTG CTG AAC AGA TAC GCC GTG GAG GGA CGA GAC CTG ACC TTG Leu Leu Ash Arg Tyr Ala Val G1u Gly Arg Asp Leu Thr Leu
176
CAG TAC AAC ATC TAC AAT GTT GGC TCA AGT GCT GCA TTA GAC Gin Tyr Asn Ile Tyr Asn Val Gly Ser Ser Ala Ala Leu Asp
218
GTG GAA CTA TCT GAT GAT TCC TTC CCT CCA GAA GAC ITT GGC Val Glu Leu Ser Asp Asp Ser Phe Pro Pro Glu Asp Phe Gly
260
ATT GIG TCT GGA ATG CIC AAT GTC AAA TGG GAC CGG ATT GCC Ile Val Ser Gly Met Leu Ash Val Lys Trp Asp Arg lle Ala
302
CCT GCT AGC AAT GTC TCC CAC ACT GTG GIC CIG CGC CCT CTC Pro Ala Ser Ash Val Ser His lhr Val Val Leu Arg Pro keu
344
AAG GCT GGT TAT ITC AAC TTC ACC TCG GCA ACA ATT ACT TAC Lys Ala Gly Tyr Phe Asn Phe lhr Ser Ala Thr lle Thr Tyr
386
CTG GCC CAG GAG GAI GGG CCC GTT GTG ATT GGC TCT ACC AGT Leu Ala Gln Glu Asp Gly Pro Val Val lle Gly Ser Thr Ser
428
GCA CCT GGA CAG GGA GGA ATC CTG GCT CAG CGG GAG TIT GAC Ala Pro Gly Gin Gly Gly lle Leu Ala Gin Arg Glu Phe Asp
470
AGG CGA TTC TCC CCI CA1 ITT CTG GAC TGG GCA GCC TIT GGG Arg Arg Phe Ser Pro His Phe Leu Asp Trp Ala Ala Phe Gly
512
GTC ATG ACC CTT CCC TCC ATC GGC ATC CCC CTG CTA TTG TGG Val Met Thr Leu Pro Ser lle Gly lle Pro Leu Leu Leu Trp
554
TAC TCC AGC AAG AGG AAA TAT GAC ACT CCC AkJ~ACG AAG AAG Tyr Ser Ser Lys Arg Lys Tyr Asp Thr Pro Lys Thr Lys Lys
596
AAC TGATTGGGGCTTCCACAGCCCTCCTCTCCCAAGAAATCCAGGCTCCTCTCC Asn > >
650
CAAG#J~ATCCAGGTGCTTTCCAGACTCCA$b~GGGTATCTTAk.ATGCk.~TCTCTTC 705 TCTCTTAGCCCTTGGCCACTTTCTCCTGGATCCTGCCCTGCTCTCAGCCATAGTG 160 AAGGACCAGCCCTAGGAGTCTGCGAGAGCCTCCTTGGTTCCATCGTGAAGCCATA 815 AACAGGAATGCCTTTGGCAATAGCCTTGAGCCTAGAGGGCCCTCTGATGCCCCAC 870 TGAGGTGCTGTTGGTTTATTGCTGGCAACGTGAATTCTCTCAGGGGTCTAGGAGG 925 GGCATTTTGGAGACTGCCTGACACCACCCCTATCCCCTGCCTCCCCCTCTCAGAA 980 GAGGGTGGAAGATGAAJ~TGAAAGCTATGGGACTCTTGGAGGATACCCAGTGTCTA 1 0 3 5 TTCTGGGTTAGAGAAGTGCTTACTAAGGGGTTTTCTAATAAAAACAAATGCCAC 1089
Fig. 1. Nucleotide sequence of the human TRAP/3 subunit cDNA and deduced amino acid sequence. The first nucleotide of each
22-bp repeat is indicated by an arrow. The polyadenylation signal is underlined. Accession number X74104.
sequence, including a stretch of hydrophobic amino acid residues and a cleavage site conforming to Von Heijne's rule [10]. The amino acid sequences of the human and canine transmembrane domains are identi]0 20 30 40 50 60 70 MRLLSFVVLALFAVTQAEEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSFPPEDFG MRLLASVLLALFAVSHAEEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSFPPEDFG +
--
+
+
+
-
+_
.
.
80 90 100 110 120 130 140 [VSGMLNVKWDRIAPASNVSHTVVLRPLKAGYFNFTSATITYLAQEDGPVVIGSTSAPGQGGI LAQREFD ..................................................................... IVSGMLNVKWDRIAPASNVSHTVVLRPLKAGYFNFTSATVTYLAQEDGPVVIGFTSAPGQGGI LAQREFD +
+
*
+
+
*
__
+_
_
150 160 170 180 RRFSPHFLDWAAFGVMTLPSIGIPLLLWYSSKRKYDTPKTKKN ....................................... RRFSPHFLDWAAFGVMTLPSIGI PLLkWYSSKRKYDTPKSKKN ++
+++
-
+
++
Fig. 2. Comparison of the amino acid sequences of the human (top) and canine (bottom) T R A P / 3 subunits. Identical amino acid residues are indicated by a double dash, conservative changes by a single dash and non-conservative changes by the absence of a dash. The signal sequence and the transmembrane domain are underlined. The glycosylated gln residues are indicated by asterisks. The positively and negatively charged amino acid residues are indicated by + and - .
cal. Secondary structure predictions indicated that the canine polypeptide contains /3 sheets adjacent to the transmembrane domain in the luminal region, which could be responsible for the interaction with the other subunits [7]. The luminal regions of the human and canine polypeptides differ by two amino acid residues, with one conservative and one non-conservative change. The canine polypeptide contains two glycosylated gln residues in the luminal region [7], which are conserved in the human polypeptide. The human and canine polypeptides show no difference with respect to their positively and negatively charged amino acid residues. The 3'-terminal nucleotide sequence of the human TRAP/3 subunit c D N A is 96% (334/348 and 422/438) homologous to the partial nucleotide sequences of two anonymous cDNAs randomly cloned from human fetal and infant brain c D N A libraries (accession numbers M78597 and T03363, respectively) [11,12]. The sense strand of the human TRAP/3 subunit c D N A sequence is homologous to the antisense strands of the anonymous sequences. This probably indicates that the anonymous cDNAs were sequenced from their poly(A) tails [12] and that the resulting sequences were recorded as such. The nucleotide sequence differences are likely to be due to sequencing errors, since only one strand of each anonymous c D N A was sequenced [12] and the number of differences increases as the distance from the poly(A) tail increases. References 1 Rapoport, T.A. (1992) Science 258, 931-936. 2 Wiedmann, M., Kurzchalia, T.V., Hartmann, E. and Rapoport, T.A. (1987) Nature 328, 830-833. 3 Hartmann, E., Wiedmann, M. and Rapoport, T.A. (1989) EMBO J. 8, 2225-2229. 4 Vogel, F., Hartmann, E., G6rlich, D. and Rapoport, T.A. (1990) Eur. J. Cell Biol. 53, 197-202. 5 Migliaccio, G., Nicchitta, C.V. and Blobel, G. (1992) J. Cell Biol. 117, 15-25. 6 Prehn, S., Herz, J., Hartmann, E., Kurzchalia, T.V., Frank, R., Roemiscb, K., Dobberstein, B. and Rapoport, T.A. (1990) Eur. J. Biochem. 188, 439-445. 7 G6rlich, D., Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B. and Rapoport, T.A. (1990)J. Cell Biol. 111, 2283-2294. 8 Hartmann, E., G6rlich, D., Kostka, S., Otto, A.~ Kraft, R., Knespel, S., Biirger, E., Rapoport, T.A. and Prehn, S. (1993) Eur. J. Biochem. 214, 375-381. 9 Kozak, M. (1989) J. Cell Biol. 108, 229-241. 10 Von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21. 11 Adams, M.D., Dubnick, M., Kerlavage, A.R., Moreno, R., Kelley, J.M., Utterbach, T.R., Nagle, J.W., Fields, C. and Venter, J.C. (1992) Nature 355, 632-634. 12 Khan, A.S., Wilcox, A.S., Polymeropoulos, M.H., Hopkins, J.A., Stevens, T.J., Robinson, M., Orpana, A.K. and Sikela, J.M. (1992) Nature Genet. 2, 180-185.