Transport and stability of the Drosophila photoreceptor proteins rhodopsin and TRPL

果蝇复眼的感光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。这些包括几个分子伴侣，护送视紫红质通过分泌途径以及Rab GTP酶和Rab相互作用蛋白。视紫红质从膜中的去除通常取决于其与arrestins的相互作用，但除此之外，依赖于视觉G蛋白的激活的arrestins独立的内化途径似乎存在。至少有两种蛋白质参与视紫红质的运输，XPORT和Rab 11，也需要运输的离子通道TRP的横纹肌感光膜，这表明TRP和视紫红质的共同运输途径。第二个光激活离子通道TRPL的运输不依赖于XPORT和Rab 11，因此似乎利用了不同的运输途径。在这个项目中，我们打算研究视紫红质和TRPL的周转机制，并确定新的视紫红质和TRPL转运途径的蛋白质。通过使用果蝇突变体表达的组成型活性Gqalpha蛋白，我们将研究拟议的G蛋白依赖性，抑制蛋白独立的视紫红质内化途径。该突变体显示视网膜变性，我们将确定变性是否是由组成性Ca 2+流入感光细胞或增强视紫红质内化引起的。为了剖析这些可能性，我们将组成型活性Gqalpha等位基因交叉到突变背景中，关闭光转导，因此Ca 2+流入Gqalpha下游，或者使用具有减少的视紫红质含量的果蝇。关于TRPL转运，我们先前已经鉴定了一种孤儿蛋白，称为CG 30118，其是TRPL转运到横纹肌所需的，但不是TRP或视紫红质的转运所需的。我们将对CG 30118进行生物化学表征，并鉴定CG 30118相互作用伙伴，以获得有关该蛋白在TRPL转运中发挥作用的线索。最后，我们打算确定新的视紫红质和TRPL相互作用的合作伙伴在一定的光照条件下，对应于不同的步骤，其营业额。这些相互作用的合作伙伴的识别将实现通过蛋白质组学方法，使用免疫共沉淀和定量质谱。该方法的可行性已经在TRPL的初步实验中进行了测试，该实验已经鉴定了可能的TRPL相互作用伴侣，例如arrestin 1，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，该项目的结果应有助于了解感光细胞中不同类型的完整质膜蛋白的主要运输途径。

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.