The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.
The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.
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Department of Zoology, University of Western Ontario, London, Canada.
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Kelly
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis*; D002645:Chickens; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism* / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005775:Gastrula / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009672:Notochord / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000378:metabolism; D021101:Radiation Hybrid Mapping; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis*; D018000:Receptors, Peptide / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D015027:Zebrafish / Q000196:embryology*; D020931:ran GTP-Binding Protein / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; C091775:ran-binding protein 1; C066973:signal sequence receptor; D020931:ran GTP-Binding Protein