Elucidating VIPP function in thylakoid biogenesis with VIPPaccumulating mutants as entry point

VIPP1（最初被称为质体中的囊泡诱导蛋白1）对类囊体膜（TMs）的生物发生很重要，因为拟南芥敲除突变体缺乏类囊体膜，而敲除突变体含有较少的TMs和TM蛋白复合物。VIPPs在暴露于高光强度的衣藻细胞和参与TM蛋白易位/整合的蛋白水平降低的突变体中过度积累（alb3.1, secA）。这表明VIPP1在TM生物发生中的作用可能不是通过囊泡形成的膜生物发生水平，而是在TM蛋白复合物的生物发生/修复水平。基于这些发现，我们对莱茵衣藻进行了正向遗传学筛选，以鉴定组成性过表达VIPPs的突变体。由于到目前为止分析的所有突变体（13个突变体中有7个）在四种主要TM蛋白复合物的亚基积累和/或光系统II的光损伤修复方面存在缺陷，这确实表明VIPPs在TM蛋白复合物的生物发生/修复中发挥了作用。在这个项目中，我们希望详细描述这些突变体，以(i)了解受影响基因在TM蛋白复合物生物发生/修复中的作用，以及（ii）了解VIPPs支持其功能的原因和方式。此外，在确定了重组VIPP与之强结合的脂质成分后，我们希望在体外和体内研究该脂质成分在VIPP膜结合和低聚化中的作用。

VIPP1 (originally termed vesicle inducing protein in plastids 1) is important for the biogenesis of thylakoid membranes (TMs), as Arabidopsis knock-out mutants lack thylakoid membranes and knockdown mutants contain fewer TMs and TM protein complexes. VIPPs overaccumulate in Chlamydomonas cells exposed to high-light intensities and in mutants with reduced levels of proteins involved in the translocation/ integration of TM proteins (alb3.1, secA). This suggests that the role of VIPP1 during TM biogenesis might not be at the level of membrane biogenesis via vesicle formation, but rather at the level of the biogenesis/repair of TM protein complexes. Based on these findings we performed a forward genetics screen with Chlamydomonas reinhardtii to identify mutants that constitutively overexpress VIPPs. As all mutants analyzed so far (7 out of 13) have defects in the accumulation of subunits of the four major TM protein complexes and/or in the repair of photosystem II from photodamage, this indeed indicates a role for VIPPs in TM protein complex biogenesis/repair. In this project we wish to characterize these mutants in detail to (i) learn about the roles of the affected genes in TM protein complex biogenesis/repair and (ii) understand why and how their functions are supported by VIPPs. Moreover, having identified a lipid component to which recombinant VIPPs strongly bind, we wish to investigate the role of this lipid component for VIPP membrane binding and oligomerization in vitro and in vivo.