Shuttling of Peroxisomal Targeting Sequence Receptors

描述(申请人提供)：过氧酶体在脂类代谢中起作用。至少有25种人类疾病会影响过氧化物酶的功能或亚细胞位置，或损害过氧化物体的生物发生。蛋白质对过氧体基质和膜的靶向是由过氧体靶向信号(PTS)介导的，其中PTS1和PTS2允许蛋白质进入基质，而MPTS促进蛋白质进入膜。参与基质蛋白输入的两个关键过氧化物酶Pex5p和Pex7p分别与胞浆中的PTS1和PTS2序列结合。在货物转运到细胞器基质之前，产生的货物-受体复合体与其他过氧化物膜上的过氧物对接。关于Pex5p和Pex7p亚细胞位置的不同结果可以用PTS受体在进口周期中从胞浆穿梭到过氧化体基质，然后返回胞浆的假设来协调。自上次提交以来，我们已经为人类Pex5p(HsPex5p)的这种扩展穿梭模型提供了证据。我们拟议的工作集中在Pex5p和Pex7p在人和酵母细胞中的进口周期中的行程，以及这个穿梭周期中各个步骤的机制细节。我们的研究将揭示这些受体的作用机制，这些受体在致命疾病中发生突变，相对于其他细胞器使用的信号序列受体是独特的。其目的是：1.HsPex5p的哪些片段允许其进出过氧酶体？HsPex5p的不同亚型会穿梭吗？HsPex5p与过氧化体停靠和易位机制的货物结合或相互作用是其穿梭所必需的吗？2.Pex5p的穿梭进出过氧化体在酵母菌中是否保守？3.Pex5p穿梭过程中的步骤，如转移到转位、膜转位、货物释放和受体输出，是什么机制？我们还将在体外重建受体穿梭。在酵母和哺乳动物中，Pex5p的穿梭进出过氧体需要哪些过氧化物素？5.Pex7p循环进出过氧酶体基质吗？如果是这样的话，Pex7p的穿梭机制是什么？

DESCRIPTION (provided by applicant): Peroxisomes play a role in lipid metabolism. At least 25 human diseases affect the function or subcellular location of peroxisomal enzymes, or impair peroxisome biogenesis. The targeting of proteins to the peroxisomal matrix and membrane is mediated by peroxisomal targeting signals (PTSs), of which PTS1 and PTS2 allow import of proteins to the matrix, and the mPTS facilitates import to the membrane. Two key peroxins involved in matrix protein import, Pex5p and Pex7p, bind the PTS1 and PTS2 sequences, respectively, in the cytosol. The resulting cargo-receptor complexes dock with other peroxins on the peroxisomal membrane, before cargo translocation occurs into the organelle matrix. The different results obtained regarding the subcellular locations of Pex5p and Pex7p can be reconciled by the hypothesis that PTS receptors shuttle from the cytosol to the peroxisome matrix and then return to the cytosol during the import cycle. We have provided evidence, since the last submission, for this extended-shuttle model for human Pex5p (HsPex5p). Our proposed work focuses on the itineraries of Pex5p and Pex7p during the import cycle in human and yeast cells, and the mechanistic details of various steps of this shuttling cycle. Our studies will reveal the mechanism of action of these receptors, which are mutated in fatal disorders and are unique relative to signal-sequence receptors used by other organelles. The aims are:1. What are the segments of HsPex5p that allow its import and export into and out of peroxisomes? Do different isoforms of HsPex5p shuttle? Are cargo binding or interactions of HsPex5p with the peroxisomal docking and translocation machinery necessary for its shuttling?2. Is the shuttling of Pex5p into and out of peroxisomes conserved in yeasts?3. What is the mechanism of steps in Pex5p shuttling such as transfer to the translocon, membrane translocation, cargo release and receptor export? We will also reconstitute receptor shuttling in vitro.4. What peroxins are necessary for the shuttling of Pex5p into and out of peroxisomes?5. Does Pex7p cycle into and out of the peroxisome matrix in yeast and in mammals? If so, what is the mechanism by which Pex7p shuttles?