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A Xenopus cysteine string protein with a cysteine residue in the J domain1
Biochimica et Biophysica Acta 1401 Ž1998. 239–241
Short sequence-paper
A Xenopus cysteine string protein with a cysteine residue in the J domain 1 Alessandro Mastrogiacomo a , Harley I. Kornblum b, Joy A. Umbach a , Cameron B. Gundersen a,) a
Department of Molecular and Medical Pharmacology, UCLA School of Medicine Los Angeles, CA 90095, USA b Department of Pediatrics, UCLA School of Medicine Los Angeles, CA 900952, USA Received 29 July 1997; revised 1 December 1997; accepted 19 December 1997
Abstract A cDNA clone encoding a Xenopus cysteine string protein ŽXcsp. was isolated and sequenced. The deduced primary sequence of Xcsp is very similar to other vertebrate csps with the exception of a cysteine residue that lies outside of the cysteine-string domain. This cysteine residue replaces a serine that is highly conserved among vertebrate csps, and thus may be of functional importance. Xcsp mRNA appears as a 4.6 kb species on Northern analysis, and immunoblot of Xenopus brain membranes reveals a single, 35 kDa Xcsp that can be deacylated, like other csps. q 1998 Elsevier Science B.V. Keywords: cDNA clone; Cysteine string protein; J domain; Ž Xenopus .
Cysteine string proteins Žcsps; w1x. are a family of secretory vesicle proteins w2,3x that have been identified in organisms as diverse as the nematode worm, Caenorhabditis elegans, and man w3,4x. The distinguishing primary sequence characteristics of csps are the cysteine-rich, cysteine-string domain and the evolutionarily conserved J domain w3x. The J domain of csps is related to the J domain of bacterial DnaJ proteins w3,5,6x. DnaJ proteins interact with Hsp70
Abbreviations: csp, cysteine string protein Corresponding author. Fax: q1-310-206-5052; E-mail: [email protected] 1 The complete nucleotide sequence of the open reading frame of Xcsp is available from EMBLrGen Bank Data Libraries under the accession number AF 015662. )
molecular chaperones in a variety of systems w5,6x, and rat csps associate with Hsp70, in vitro w7,8x. However, the purpose of this presumed csp–Hsp70 interaction in vivo remains unknown. As part of an effort to learn more about the physiological role of csps including their interaction with Hsp70, we have begun to exploit a system in which Xenopus motor neurons form synapses with muscle cells in culture w9x. The synapses formed by these embryonic cells are amenable to various types of investigations that are germane to understanding synaptic transmission w10,11x. To facilitate our planned studies, it was essential first to clone and sequence cDNA for Xcsp. Anti-csp antibodies were used Žas in Ref. w4x. to screen a lZap expression library constructed from RNA of dorsal lips of Xenopus laeÕis gastrulae w12x.
0167-4889r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 1 6 0 - 2
240
A. Mastrogiacomo et al.r Biochimica et Biophysica Acta 1401 (1998) 239–241
A single positive clone was identified with an insert of about 3.5 kb. Nucleotide sequencing revealed that this clone harbored the full open reading frame of Xcsp preceded by about 80 nucleotides of 5X noncoding region. The deduced amino acid sequence of Xcsp is in Fig. 1A. The cysteine-string domain is underlined, and the unusual cysteine residue at position 34 is boxed Ž Fig. 1A. . The alignment of vertebrate csps ŽFig. 1B. shows that this cysteine residue replaces a highly conserved serine found in all other
Fig. 2. Northern analysis of adult Xenopus brain poly ŽA.q RNA. Poly ŽA.q RNA was isolated from a single Xenopus brain using the Quick-prep kit ŽPharmacia. and resolved electrophoretically for high-stringency Northern analysis as in Ref. w4x. Gel-purified Sty I fragments of Xcsp plasmid DNA were used to probe this Northern. Immunoblot analysis of Xenopus brain membranes. A sample Ž50 m g protein. of the P2 fraction of Xenopus brain was either used directly or it was pre-treated with deacylating reagent Ž1 M hydroxylamine, pH 7.0. prior to immunoblot analysis as described in Ref. w4x. Protein standards ŽAmersham. were used to calibrate the mass of the immunoreactive species. Without hydroxylamine ŽyNH 2 OH. Xcsp migrates at about 35 kDa whereas after deacylation ŽqNH 2 OH. it runs as a 28 kDa species.
Fig. 1. ŽA. The deduced primary sequence of Xcsp. The singleletter code is used. The cysteine string is underlined, while the lone cysteine residue in the J domain is boxed. ŽB. Alignment of deduced primary sequences of vertebrate csps. The J domains Žresidues 1–70. are underlined while the cysteine strings have bold underlines. The unusual cysteine Ž34. in Xcsp is bold and underlined. The prefixes denote csps of human ŽH.; bovine ŽB.; rat ŽR. and Torpedo ŽT. origin as accessed via Genbank.
vertebrate csps. wTo verify the identity of this cysteine residue, we sequenced two independent PCR-derived fragments of Xcsp genomic DNAx. Aside from this Cys for Ser substitution there is a high degree of sequence conservation among csps ŽFig. 1B. . Overall, Xcsp is 85% identical to Torpedo Žfish. csp and 86% identical to rat csp. Northern analysis of adult Xenopus brain poly ŽA.q RNA reveals a single hybridizing species of about 4.6 kb ŽFig. 2A.. Since the 3X end of the Xcsp cDNA clone includes part of the poly A tail Ž data not shown, but see Ref. w12x., we infer from the location of the Xcsp open reading frame that Xcsp mRNA has approximately 1.2 kb of 5X non-coding sequence and about 2.8 kb of 3X noncoding RNA. Immunoreactive Xcsp in Xenopus brain migrates as a 35 kDa species prior to deacylation and as a 28 kDa species after deacylation Ž Fig. 2B. . This mass shift is typical of csps in other species w3,4,13x, but this procedure cannot tell us whether cysteine 34 is fatty acylated in Xcsp.
A. Mastrogiacomo et al.r Biochimica et Biophysica Acta 1401 (1998) 239–241
The deduced primary sequence of Xcsp will facilitate various studies of the role of this protein in transmitter secretion at Xenopus nerve-muscle synapses in culture. For instance, the design of prospective peptide perturbants of csp function demands that we know the sequence of Xcsp. Moreover, the finding that Xcsp has a cysteine residue in the J domain has several interesting ramifications. First, if this cysteine residue is fatty acylated Ž and the majority of the cysteine residues of csps are fatty acylated; w3,13x, it is likely that the addition of such a hydrophobic moiety to the J domain of csp would affect its interaction with Hsp70 in vivo. Second, if this cysteine escapes fatty acylation, then Xcsp may be useful in studies of the mechanisms governing the selective fatty acylation of cysteine residues in this and other proteins. Third, this cysteine replaces a serine residue that is highly conserved among vertebrate csps ŽFig. 1B. where it is a candidate for phosphorylation by C kinase. This result suggests that regulation of Xcsp may differ from that of other vertebrate csps. Thus, we conclude that future studies of csps and their post-translational modification in Xenopus will offer valuable insights into secretory events at nerve terminals and elsewhere. We thank Dr. Eddy DeRobertis for the cDNA library, Mike Phelps for support, and Theresa Sama for preparing the manuscript. Supported in part by a grant from NIH ŽNS 31934. to Joy Umbach; a DOE
241
cooperative agreement, and the UCLA Child Health Research Center ŽP30 HD34610..
References w1x K. Zinsmaier, A. Hofbauer, G. Heimbeck, G. Pflugfelder, S. Buchner, E. Buchner, J. Neurogenet. 7 Ž1990. 15–29. w2x A. Mastrogiacomo, S.M. Parsons, G.A. Zampighi, D.J. Jenden, J.A. Umbach, C.B. Gundersen, Science 263 Ž1994. 981–982. w3x E. Buchner, C.B. Gundersen, TINS 20 Ž1997. 223–228. w4x T. Coppola, C.B. Gundersen, FEBS Lett. 391 Ž1996. 269– 272. w5x A.J. Caplan, D.M. Cyr, M.G. Douglas, Mol. Cell. Biol. 4 Ž1993. 555–563. w6x P.A. Silver, J.C. Way, Cell 74 Ž1993. 5–6. w7x L.H. Chamberlain, R.D. Burgoyne, Biochem. J. 322 Ž1997. 853–858. w8x J.E.A. Braun, S.M. Wilbanks, R.H. Scheller, J. Biol. Chem. 271 Ž1996. 25989–25993. w9x R.E. Poage, S.D. Meriney, A. Mastrogiacomo, C.B. Gundersen, J.A. Umbach, Neurosci. Abs. 23 Ž1997. 578. w10x J. Alder, Z.-P. Xie, F. Valtorta, P. Greengard, M. Poo, Neuron 9 Ž1992. 759–768. w11x B. Yazejian, D.A. DiGregorio, J.L. Vergara, R.E. Poage, S.D. Meriney, A.D. Grinnell, J. Neurosci. 17 Ž1997. 2990– 3001. w12x B. Blumberg, C.V.E. Wright, E.M. DeRobertis, K.W.Y. Cho, Science 253 Ž1991. 194–196. w13x C.B. Gundersen, A. Mastrogiacomo, K. Faull, J.A. Umbach, J. Biol. Chem. 269 Ž1994. 19197–19199.
Short sequence-paper
A Xenopus cysteine string protein with a cysteine residue in the J domain 1 Alessandro Mastrogiacomo a , Harley I. Kornblum b, Joy A. Umbach a , Cameron B. Gundersen a,) a
Department of Molecular and Medical Pharmacology, UCLA School of Medicine Los Angeles, CA 90095, USA b Department of Pediatrics, UCLA School of Medicine Los Angeles, CA 900952, USA Received 29 July 1997; revised 1 December 1997; accepted 19 December 1997
Abstract A cDNA clone encoding a Xenopus cysteine string protein ŽXcsp. was isolated and sequenced. The deduced primary sequence of Xcsp is very similar to other vertebrate csps with the exception of a cysteine residue that lies outside of the cysteine-string domain. This cysteine residue replaces a serine that is highly conserved among vertebrate csps, and thus may be of functional importance. Xcsp mRNA appears as a 4.6 kb species on Northern analysis, and immunoblot of Xenopus brain membranes reveals a single, 35 kDa Xcsp that can be deacylated, like other csps. q 1998 Elsevier Science B.V. Keywords: cDNA clone; Cysteine string protein; J domain; Ž Xenopus .
Cysteine string proteins Žcsps; w1x. are a family of secretory vesicle proteins w2,3x that have been identified in organisms as diverse as the nematode worm, Caenorhabditis elegans, and man w3,4x. The distinguishing primary sequence characteristics of csps are the cysteine-rich, cysteine-string domain and the evolutionarily conserved J domain w3x. The J domain of csps is related to the J domain of bacterial DnaJ proteins w3,5,6x. DnaJ proteins interact with Hsp70
Abbreviations: csp, cysteine string protein Corresponding author. Fax: q1-310-206-5052; E-mail: [email protected] 1 The complete nucleotide sequence of the open reading frame of Xcsp is available from EMBLrGen Bank Data Libraries under the accession number AF 015662. )
molecular chaperones in a variety of systems w5,6x, and rat csps associate with Hsp70, in vitro w7,8x. However, the purpose of this presumed csp–Hsp70 interaction in vivo remains unknown. As part of an effort to learn more about the physiological role of csps including their interaction with Hsp70, we have begun to exploit a system in which Xenopus motor neurons form synapses with muscle cells in culture w9x. The synapses formed by these embryonic cells are amenable to various types of investigations that are germane to understanding synaptic transmission w10,11x. To facilitate our planned studies, it was essential first to clone and sequence cDNA for Xcsp. Anti-csp antibodies were used Žas in Ref. w4x. to screen a lZap expression library constructed from RNA of dorsal lips of Xenopus laeÕis gastrulae w12x.
0167-4889r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 1 6 0 - 2
240
A. Mastrogiacomo et al.r Biochimica et Biophysica Acta 1401 (1998) 239–241
A single positive clone was identified with an insert of about 3.5 kb. Nucleotide sequencing revealed that this clone harbored the full open reading frame of Xcsp preceded by about 80 nucleotides of 5X noncoding region. The deduced amino acid sequence of Xcsp is in Fig. 1A. The cysteine-string domain is underlined, and the unusual cysteine residue at position 34 is boxed Ž Fig. 1A. . The alignment of vertebrate csps ŽFig. 1B. shows that this cysteine residue replaces a highly conserved serine found in all other
Fig. 2. Northern analysis of adult Xenopus brain poly ŽA.q RNA. Poly ŽA.q RNA was isolated from a single Xenopus brain using the Quick-prep kit ŽPharmacia. and resolved electrophoretically for high-stringency Northern analysis as in Ref. w4x. Gel-purified Sty I fragments of Xcsp plasmid DNA were used to probe this Northern. Immunoblot analysis of Xenopus brain membranes. A sample Ž50 m g protein. of the P2 fraction of Xenopus brain was either used directly or it was pre-treated with deacylating reagent Ž1 M hydroxylamine, pH 7.0. prior to immunoblot analysis as described in Ref. w4x. Protein standards ŽAmersham. were used to calibrate the mass of the immunoreactive species. Without hydroxylamine ŽyNH 2 OH. Xcsp migrates at about 35 kDa whereas after deacylation ŽqNH 2 OH. it runs as a 28 kDa species.
Fig. 1. ŽA. The deduced primary sequence of Xcsp. The singleletter code is used. The cysteine string is underlined, while the lone cysteine residue in the J domain is boxed. ŽB. Alignment of deduced primary sequences of vertebrate csps. The J domains Žresidues 1–70. are underlined while the cysteine strings have bold underlines. The unusual cysteine Ž34. in Xcsp is bold and underlined. The prefixes denote csps of human ŽH.; bovine ŽB.; rat ŽR. and Torpedo ŽT. origin as accessed via Genbank.
vertebrate csps. wTo verify the identity of this cysteine residue, we sequenced two independent PCR-derived fragments of Xcsp genomic DNAx. Aside from this Cys for Ser substitution there is a high degree of sequence conservation among csps ŽFig. 1B. . Overall, Xcsp is 85% identical to Torpedo Žfish. csp and 86% identical to rat csp. Northern analysis of adult Xenopus brain poly ŽA.q RNA reveals a single hybridizing species of about 4.6 kb ŽFig. 2A.. Since the 3X end of the Xcsp cDNA clone includes part of the poly A tail Ž data not shown, but see Ref. w12x., we infer from the location of the Xcsp open reading frame that Xcsp mRNA has approximately 1.2 kb of 5X non-coding sequence and about 2.8 kb of 3X noncoding RNA. Immunoreactive Xcsp in Xenopus brain migrates as a 35 kDa species prior to deacylation and as a 28 kDa species after deacylation Ž Fig. 2B. . This mass shift is typical of csps in other species w3,4,13x, but this procedure cannot tell us whether cysteine 34 is fatty acylated in Xcsp.
A. Mastrogiacomo et al.r Biochimica et Biophysica Acta 1401 (1998) 239–241
The deduced primary sequence of Xcsp will facilitate various studies of the role of this protein in transmitter secretion at Xenopus nerve-muscle synapses in culture. For instance, the design of prospective peptide perturbants of csp function demands that we know the sequence of Xcsp. Moreover, the finding that Xcsp has a cysteine residue in the J domain has several interesting ramifications. First, if this cysteine residue is fatty acylated Ž and the majority of the cysteine residues of csps are fatty acylated; w3,13x, it is likely that the addition of such a hydrophobic moiety to the J domain of csp would affect its interaction with Hsp70 in vivo. Second, if this cysteine escapes fatty acylation, then Xcsp may be useful in studies of the mechanisms governing the selective fatty acylation of cysteine residues in this and other proteins. Third, this cysteine replaces a serine residue that is highly conserved among vertebrate csps ŽFig. 1B. where it is a candidate for phosphorylation by C kinase. This result suggests that regulation of Xcsp may differ from that of other vertebrate csps. Thus, we conclude that future studies of csps and their post-translational modification in Xenopus will offer valuable insights into secretory events at nerve terminals and elsewhere. We thank Dr. Eddy DeRobertis for the cDNA library, Mike Phelps for support, and Theresa Sama for preparing the manuscript. Supported in part by a grant from NIH ŽNS 31934. to Joy Umbach; a DOE
241
cooperative agreement, and the UCLA Child Health Research Center ŽP30 HD34610..
References w1x K. Zinsmaier, A. Hofbauer, G. Heimbeck, G. Pflugfelder, S. Buchner, E. Buchner, J. Neurogenet. 7 Ž1990. 15–29. w2x A. Mastrogiacomo, S.M. Parsons, G.A. Zampighi, D.J. Jenden, J.A. Umbach, C.B. Gundersen, Science 263 Ž1994. 981–982. w3x E. Buchner, C.B. Gundersen, TINS 20 Ž1997. 223–228. w4x T. Coppola, C.B. Gundersen, FEBS Lett. 391 Ž1996. 269– 272. w5x A.J. Caplan, D.M. Cyr, M.G. Douglas, Mol. Cell. Biol. 4 Ž1993. 555–563. w6x P.A. Silver, J.C. Way, Cell 74 Ž1993. 5–6. w7x L.H. Chamberlain, R.D. Burgoyne, Biochem. J. 322 Ž1997. 853–858. w8x J.E.A. Braun, S.M. Wilbanks, R.H. Scheller, J. Biol. Chem. 271 Ž1996. 25989–25993. w9x R.E. Poage, S.D. Meriney, A. Mastrogiacomo, C.B. Gundersen, J.A. Umbach, Neurosci. Abs. 23 Ž1997. 578. w10x J. Alder, Z.-P. Xie, F. Valtorta, P. Greengard, M. Poo, Neuron 9 Ž1992. 759–768. w11x B. Yazejian, D.A. DiGregorio, J.L. Vergara, R.E. Poage, S.D. Meriney, A.D. Grinnell, J. Neurosci. 17 Ž1997. 2990– 3001. w12x B. Blumberg, C.V.E. Wright, E.M. DeRobertis, K.W.Y. Cho, Science 253 Ž1991. 194–196. w13x C.B. Gundersen, A. Mastrogiacomo, K. Faull, J.A. Umbach, J. Biol. Chem. 269 Ž1994. 19197–19199.