Wagner Stefanie, Heseding Christiane, Szlachta Kamila, True John R, Prinz Heino, Hovemann Bernhard T
Fakultät für Chemie, AG Molekulare Zellbiochemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
J Comp Neurol. 2007 Feb 1;500(4):601-11. doi: 10.1002/cne.21138.
The Drosophila mutant tan (t) shows reciprocal pigmentation defects compared with the ebony (e) mutant. Visual phenotypes, however, are similar in both flies: Electroretinogram (ERG) recordings lack "on" and "off" transients, an indication of impaired synaptic transmission to postsynaptic cells L1 and L2. Cloning of tan revealed transcription of the gene in the retina, apparently in photoreceptor cells. We expressed Tan in Escherichia coli and confirmed by Western blotting and mass spectroscopic analyses that Tan is expressed as preprotein, followed by proteolytic cleavage into two subunits at a conserved --Gly--Cys-- motif like its fungal ortholog isopenicillin-N N-acyltransferase (IAT). Tan thus belongs to the large family of cysteine peptidases. To discriminate expression of Tan and Ebony in retina and optic neuropils, we raised antisera against specific Tan peptides. Testing for colocalization with GMR-driven n-Syb-GFP labeling revealed that Tan expression is confined to the photoreceptor cells R1-R8. A close proximity of Tan and Ebony expression is evident in lamina cartridges, where three epithelial glia cells envelop the six photoreceptor terminals R1-R6. In the medulla, R7/R8 axonal terminals appeared lined up side by side with glial extensions. This local proximity supports a model for Drosophila visual synaptic transmission in which Tan and Ebony interact biochemically in a putative histamine inactivation and recycling pathway in Drosophila.
果蝇突变体棕褐色(tan,t)与乌木色(ebony,e)突变体相比表现出相反的色素沉着缺陷。然而,两种果蝇的视觉表型相似:视网膜电图(ERG)记录缺乏“开”和“关”瞬变,这表明向突触后细胞L1和L2的突触传递受损。tan的克隆揭示了该基因在视网膜中的转录,显然是在光感受器细胞中。我们在大肠杆菌中表达了Tan,并通过蛋白质免疫印迹和质谱分析证实Tan以前体蛋白形式表达,随后在一个保守的-Gly-Cys-基序处被蛋白水解切割成两个亚基,就像其真菌同源物异青霉素-N N-酰基转移酶(IAT)一样。因此,Tan属于半胱氨酸肽酶的大家族。为了区分Tan和乌木色在视网膜和视神经节中的表达,我们制备了针对特定Tan肽的抗血清。与GMR驱动的n-Syb-GFP标记共定位测试表明,Tan的表达局限于光感受器细胞R1-R8。在小眼体中,Tan和乌木色的表达紧密相邻,其中三个上皮神经胶质细胞包裹着六个光感受器末端R1-R6。在髓质中,R7/R8轴突末端似乎与神经胶质细胞的延伸部分并排排列。这种局部相邻支持了一种果蝇视觉突触传递模型,其中Tan和乌木色在果蝇假定的组胺失活和再循环途径中发生生化相互作用。