果蝇中的线粒体天冬氨酸/谷氨酸载体（AGC 或 Aralar1）同工型：生化特性、基因结构和进化分析。

The mitochondrial aspartate/glutamate carrier (AGC or Aralar1) isoforms in D. melanogaster: biochemical characterization, gene structure, and evolutionary analysis.

机构信息

Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

Department of Biology, University of Bari, 70125 Bari, Italy.

出版信息

Biochim Biophys Acta Gen Subj. 2021 May;1865(5):129854. doi: 10.1016/j.bbagen.2021.129854. Epub 2021 Jan 23.

DOI:10.1016/j.bbagen.2021.129854

PMID:33497735

Abstract

BACKGROUND

In man two mitochondrial aspartate/glutamate carrier (AGC) isoforms, known as aralar and citrin, are required to accomplish several metabolic pathways. In order to fill the existing gap of knowledge in Drosophila melanogaster, we have studied aralar1 gene, orthologue of human AGC-encoding genes in this organism.

METHODS

The blastp algorithm and the "reciprocal best hit" approach have been used to identify the human orthologue of AGCs in Drosophilidae and non-Drosophilidae. Aralar1 proteins have been overexpressed in Escherichia coli and functionally reconstituted into liposomes for transport assays.

RESULTS

The transcriptional organization of aralar1 comprises six isoforms, three constitutively expressed (aralar1-RA, RD and RF), and the remaining three distributed during the development or in different tissues (aralar1-RB, RC and RE). Aralar1-PA and Aralar1-PE, representative of all isoforms, have been biochemically characterized. Recombinant Aralar1-PA and Aralar1-PE proteins share similar efficiency to exchange glutamate against aspartate, and same substrate affinities than the human isoforms. Interestingly, although Aralar1-PA and Aralar1-PE diverge only in their EF-hand 8, they greatly differ in their specific activities and substrate specificity.

CONCLUSIONS

The tight regulation of aralar1 transcripts expression and the high request of aspartate and glutamate during early embryogenesis suggest a crucial role of Aralar1 in this Drosophila developmental stage. Furthermore, biochemical characterization and calcium sensitivity have identified Aralar1-PA and Aralar1-PE as the human aralar and citrin counterparts, respectively.

GENERAL SIGNIFICANCE

The functional characterization of the fruit fly mitochondrial AGC transporter represents a crucial step toward a complete understanding of the metabolic events acting during early embryogenesis.

摘要

背景

在人类中，两种线粒体天冬氨酸/谷氨酸载体（AGC）同工型，即 aralar 和 citrin，需要完成几种代谢途径。为了填补果蝇（Drosophila melanogaster）中现有的知识空白，我们研究了该生物中 aralar1 基因，即人类 AGC 编码基因的同源物。

方法

使用 blastp 算法和“相互最佳命中”方法，在果蝇科和非果蝇科中鉴定出 AGC 的人类同源物。在大肠杆菌中过表达 aralar1 蛋白，并将其功能重建到脂质体中进行转运测定。

结果

aralar1 的转录组织包括六种同工型，三种组成型表达（aralar1-RA、RD 和 RF），其余三种分布在发育过程中或不同组织中（aralar1-RB、RC 和 RE）。aralar1-PA 和 aralar1-PE，代表所有同工型，已进行了生化表征。重组 Aralar1-PA 和 Aralar1-PE 蛋白在交换谷氨酸对天冬氨酸方面具有相似的效率，并且与人类同工型具有相同的底物亲和力。有趣的是，尽管 Aralar1-PA 和 Aralar1-PE 仅在其 EF 手 8 中存在差异，但它们在特定活性和底物特异性方面存在很大差异。