Assembly of the Peroxisomal Translocon

过氧化体转运蛋白与基质蛋白的组装是一个未知的多步骤过程，它由胞质受体和完全折叠的货物蛋白之间的相互作用启动。与膜蛋白Pex14p一起，受体Pex5p和共受体Pex18p分别成为PTS1或PTS2特异性输入通道的组成部分。已有研究表明，转座子的组装主要是由受体与过氧磷脂和Pex14p的相互作用驱动的，可以同时进行，也可以顺序进行。在这个项目的第一个资助期内，我们专注于受体与各种货物蛋白、PEX14和磷脂的相互作用的表征。此外，我们建立了方法，允许以空间和时间的方式剖析转位基因组装的单个步骤。在下一阶段，我们将具体解决以下目标：A.分析磷脂结合的PTS受体对转运子组装的作用B。阐明PEX14在孔道形成中的作用PEX19在转位蛋白组装中的作用在本项目的第一部分中，我们将定位人PTS1受体的磷脂相互作用部位，并利用核磁共振技术研究这对其构象、动力学和分子相互作用的影响。这些位点对组装功能孔的贡献将通过诱变这些位点并插入允许电生理孔测量的水平脂双层(HLB)来评估。脂质相互作用的生理作用也将通过在PEX5缺陷的人类细胞中表达PEX5变体来验证。对于该项目的第二部分，将采用类似的实验策略来研究所有已知的PEX5-PEX14相互作用位点在体外和体内使用定点突变的作用。这包括N-末端区域的WxxxF/Y基序和PEX5中的C-末端结合位点，这些都是在本资助期内发现的。PEX19是过氧化物体膜蛋白的受体，以PEX5竞争的方式与PEX14结合。在项目的第三部分，将结合HLB和核磁共振研究来研究PEX19对转运子组装的体外影响。这些分析可以为基质蛋白和膜蛋白的输入途径的趋同提供机械性的见解。该项目的第四部分将解决PTS受体形成的孔是否可以转移蛋白质以外的其他分子，并在此背景下与折叠的酶相关的问题。虽然已经证实了过氧化体膜上存在特定的代谢物转运体，但到目前为止还没有确定它们的存在。酵母过氧化体膜上的Pex5p亚复合体是证明这一假设的理想候选者。

The assembly of peroxisomal translocons for matrix proteins is an unknown multi-step process, which is initiated by the interaction between cytosolic receptors and fully folded cargo-proteins. Together with the membrane protein Pex14p, the receptor Pex5p and the co-receptor Pex18p become integral constituents of PTS1- or PTS2-specific import channels, respectively. It has been suggested that the assembly of translocons is mainly driven by interaction of receptors with peroxisomal phospholipids and Pex14p, either simultaneously or sequentially. Within the first funding period of this project, we focussed on the characterization of receptor interactions with various cargo-proteins, PEX14 and phospholipids. Moreover, we established methods which allow to dissect the single steps of translocon assembly in a spatial and temporal manner. In the next period, we will specifically address the following goals: A. Analysis of the role of phospholipid binding of PTS receptors for translocon assemblyB. Elucidation of the function of PEX14 for pore formationC. Role of PEX19 for translocon assemblyD. Investigation of a role of the peroxisomal PTS1 translocon in metabolite transport In the first part of the project, we will map phospholipid-interacting sites of the human PTS1 receptor and study how this affects its conformation, dynamics and molecular interactions by NMR. The contribution of these sites for the assembly of functional pores will be assessed by mutagenizing these sites and insertion into horizontal lipid bilayers (HLB) allowing electrophysiological pore measurements. The physiological role of the lipid interaction will also be validated by expressing PEX5 variants in PEX5-deficient human cells. For the second part of the project, a similar experimental strategy will be employed to study the role of all known PEX5-PEX14 interacting sites using site-directed mutagenesis in vitro and in vivo. This includes the WxxxF/Y motifs in the N-terminal region and the C-terminal binding site in PEX5, which have been identified in this funding period. PEX19, the receptor for peroxisomal membrane proteins, binds to PEX14 in a PEX5-competitive way. In the third part of the project, the in vitro effects of PEX19 on translocon assembly will be studied by combining HLB- and NMR-studies. These analyses could provide mechanistic insights into the convergence of import pathways for matrix and membrane proteins. The fourth part of the project will address the question whether the pores formed by PTS receptors can translocate other molecules than proteins and in this context remain associated with the folded enzymes. Although specific metabolite transporters in the peroxisomal membrane have been proven to exist, they could not be identified so far. A Pex5p subcomplex at the peroxisomal membrane of yeast is an ideal candidate to prove this assumption.