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The translocon-associated protein β in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors

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Abstract

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 TRAPβ subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPβ subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPβ mRNA is expressed in the developing zebrafish embryo. TRAPβ 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 TRAPβ 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 TRAPβ 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. TRAPβ, 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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Authors and Affiliations

  1. Department of Zoology, Molecular Genetics Unit, University of Western Ontario, London, On, Canada, N6A 5B7

    Steve Mangos & Roman Krawetz

  2. Department of Zoology, Molecular Genetics Unit and Department of Paediatrics, University of Western Ontario, London, On, Canada, N6A 5B7

    Gregory M. Kelly

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  1. Steve Mangos
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  2. Roman Krawetz
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  3. Gregory M. Kelly
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Mangos, S., Krawetz, R. & Kelly, G.M. The translocon-associated protein β in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors. Mol Cell Biochem 215, 93–101 (2000). https://doi.org/10.1023/A:1026598516681

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