RESUMEN
Protein transport between the trans-Golgi network and endosomes is mediated by transport vesicles formed by the adaptor-protein complex AP-1, consisting of the adaptins γ1, ß1, µ1, σ1. Mammalia express µ1A ubiquitously and isoform µ1B in polarized epithelia. Mouse γ1 or µ1A 'knock out's revealed that AP-1 is indispensable for embryonic development. We isolated µ1A and µ1B from Danio rerio. Analysis of µ1A and µ1B expression revealed tissue-specific expression for either one during embryogenesis and in adult tissues in contrast to their expression in mammalia. µ1B transcript was detected in organs of endodermal derivation and "knock-down" experiments gave rise to embryos defective in formation of intestine, liver, and pronephric ducts. Development ceased at 7-8 dpf. µ1B is not expressed in murine liver, indicating loss of µ1B expression and establishment of alternative sorting mechanisms during mammalian development.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Morfogénesis/fisiología , Isoformas de Proteínas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Complejo 1 de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/clasificación , Subunidades mu de Complejo de Proteína Adaptadora/genética , Secuencia de Aminoácidos , Animales , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genoma , Humanos , Ratones , Datos de Secuencia Molecular , Fenotipo , Filogenia , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Alineación de Secuencia , Distribución Tisular , Pez Cebra/anatomía & histología , Pez Cebra/genética , Proteínas de Pez Cebra/clasificación , Proteínas de Pez Cebra/genéticaRESUMEN
The cDNA and genomic DNA for the mu3A subunit of the AP-3 (adaptor protein-3) complex were cloned from Chinese hamster cells. The AP-3 mu3A genes in Chinese hamster, human and mouse each comprise nine exons and eight introns, with all introns located in identical positions in the species studied. The AP-3 mu3A genes in these species are linked in a head-to-head fashion with the gene for the purine salvage pathway enzyme AK (adenosine kinase). These genes share the first exon, and a 512 bp fragment covering the intervening untranslated sequence has the characteristic of a CpG island promoter, and it effectively carried out transcription in both directions. Deletion studies indicate that this region contains both positive and negative regulatory elements affecting transcription of these genes. In comparison with the AP-3 mu3A gene (27 kb), the AK gene in human is very large (558 kb), with average exon and intron lengths of approx. 100 bp and 55.7 kb respectively. The ratio of non-coding to coding sequence in the human AK gene is >550, which is the highest reported for any gene. We also present evidence that a number of AK- mutants of Chinese hamster ovary cells contain large deletions that affect both of these genes. In addition to lacking part of the AK gene, two of these mutants also lacked all of the exons and introns corresponding to the AP-3 mu3A gene. These mutants should prove useful in elucidating the role of AP-3 mu3A in vesicle-mediated protein sorting--a process that is altered in Hermansky-Pudlak syndrome. Detailed phylogenetic analysis of the micro family of proteins presented here also provides insight into how different AP complexes are related and may have evolved.