Transport and stability of the Drosophila photoreceptor proteins rhodopsin and TRPL

果蝇复眼的光感受器细胞为研究膜蛋白的转运提供了一个模型系统，视色素视紫红质转运途径的主要成分已经被确定。其中包括护送视紫质通过分泌途径的几个伴侣蛋白，以及Rab gtp酶和Rab相互作用蛋白。视紫红质从膜上的去除通常依赖于其与抑制蛋白的相互作用，但此外，依赖于视觉g蛋白激活的不依赖于抑制蛋白的内化途径似乎存在。至少有两种参与视紫红质运输的蛋白，XPORT和Rab11，也需要离子通道TRP运输到横纹肌光感受器膜，这表明TRP和视紫红质有一个共同的运输途径。第二个光激活离子通道TRPL的传输不依赖于XPORT和Rab11，因此似乎利用了一个独特的传输路线。在该项目中，我们打算研究视紫红质和TRPL转换的机制，并确定视紫红质和TRPL运输途径的新蛋白。通过使用表达组成活性gqα蛋白的果蝇突变体，我们将研究所提出的g蛋白依赖的、不依赖于抑制蛋白的视紫红质内化途径。该突变体显示视网膜变性，我们将确定变性是由组成型Ca2+内流到光感受器细胞还是由视紫红质内化增强引起的。为了分析这些可能性，我们要么将构成活性的Gqalpha等位基因交叉到关闭光转导的突变背景中，从而关闭Gqalpha下游的Ca2+内流，要么使用视紫质含量降低的果蝇。关于TRPL运输，我们之前已经发现了一种孤儿蛋白，称为CG30118，它是TRPL运输到横纹肌所必需的，但不是TRP或视紫红质的运输。我们将对CG30118进行生化表征，并鉴定CG30118的相互作用伙伴，以了解该蛋白在TRPL转运中的作用背景。最后，我们打算在特定的光照条件下确定新的视紫红质和TRPL相互作用伙伴，这些相互作用伙伴对应于它们转换的不同步骤。这些相互作用伙伴的鉴定将通过使用免疫共沉淀和定量质谱的蛋白质组学方法来实现。该方法的可行性已在TRPL的初步实验中得到验证，并已鉴定出可能的TRPL相互作用伙伴，如arrestin1、Synaptotagmin和14-3-3蛋白。现在将研究这些蛋白质在TRPL运输中的作用。总的来说，该项目的结果应该有助于理解不同类型的整体质膜蛋白在光感受器细胞中的主要运输途径。

Photoreceptor cells of the Drosophila compound eye provide a model system for studying transport of membrane proteins and major components of the transport pathways for the visual pigment rhodopsin have already been identified. These include several chaperones that escort rhodopsin through the secretory pathway as well as Rab GTPases and Rab-interacting proteins. Removal of rhodopsin from membranes usually depends on its interaction with arrestins but, in addition, an arrestin-independent internalization pathway depending on activation of the visual G-protein seems to exist. At least two of the proteins involved in rhodopsin transport, XPORT and Rab11, are also needed for transport of the ion channel TRP to the rhabdomeral photoreceptor membrane, suggesting a common transport pathway for TRP and rhodopsin. Transport of a second light-activated ion channel, TRPL, does not depend on XPORT and Rab11 and thus seems to utilize a distinct transport route. In the proposed, project we intend to study the mechanisms underlying turnover of rhodopsin and TRPL and to identify new proteins of the rhodopsin and TRPL transport routes. By using a Drosophila mutant expressing a constitutively active Gqalpha protein, we will study the proposed G-protein-dependent, arrestin-independent rhodopsin internalization pathway. This mutant shows retinal degeneration and we will determine whether degeneration results from constitutive Ca2+ influx into photoreceptor cells or from enhanced rhodopsin internalization. To dissect these possibilities, we will either cross the constitutively active Gqalpha allele into mutant backgrounds that shut down phototransduction and hence Ca2+ influx downstream of Gqalpha or use flies that have a reduced rhodopsin content. With respect to TRPL transport, we have previously identified an orphan protein, termed CG30118, that is required for TRPL transport to the rhabdomere but not for transport of TRP or rhodopsin. We will characterize CG30118 biochemically and identify CG30118 interaction partners in order to get a clue on the context in which this protein functions in the transport of TRPL. Finally, we intend to identify new rhodopsin and TRPL interaction partners at certain light conditions that correspond to distinct steps of their turnover. Identification of these interaction partners will be achieved by a proteomics approach using co-immunoprecipitation and quantitative mass spectrometry. The feasibility of this method has been tested in preliminary experiments with TRPL that already resulted in identification of possible TRPL interaction partners, e.g. arrestin1, Synaptotagmin and 14-3-3 proteins. A role of these proteins in TRPL transport will now be studied. Collectively, the results of the project should contribute to the understanding of principle transport pathways for different types of integral plasma membrane proteins in photoreceptor cells.