Abstract：
Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) acts as a negative regulator of autophagy by interacting with Beclin 1 at Golgi membranes in mammalian cells. The molecular mechanism of this interaction is largely unknown. We recently showed that human GAPR-1 (hGAPR-1) has amyloidogenic properties resulting in the formation of protein condensates upon overexpression in Saccharomyces cerevisiae. Here we show that human Beclin 1 (hBeclin 1) has several predicted amyloidogenic regions and that overexpression of hBeclin 1-mCherry in yeast also results in the formation of fluorescent protein condensates. Surprisingly, co-expression of hGAPR-1-GFP and hBeclin 1-mCherry results in a strong reduction of hBeclin 1 condensates. Mutations of the known interaction site on the hGAPR-1 and hBeclin 1 surface abolished the effect on condensate formation during co-expression without affecting the condensate formation properties of the individual proteins. Similarly, a hBeclin 1-derived B18 peptide that is known to bind hGAPR-1 and to interfere with the interaction between hGAPR-1 and hBeclin 1, abolished the reduction of hBeclin 1 condensates by co-expression of hGAPR-1. These results indicate that the same type of protein-protein interactions interfere with condensate formation during co-expression of hGAPR-1 and hBeclin 1 as previously described for their interaction at Golgi membranes. The amyloidogenic properties of the B18 peptide were, however, important for the interaction with hGAPR-1, as mutant peptides with reduced amyloidogenic properties also showed reduced interaction with hGAPR-1 and reduced interference with hGAPR-1/hBeclin 1 condensate formation. We propose that amyloidogenic interactions take place between hGAPR-1 and hBeclin 1 prior to condensate formation.
摘要：
高尔基体相关植物发病机制相关蛋白1（GAPR-1）通过与哺乳动物细胞高尔基体膜上的Beclin1相互作用，充当自噬的负调节因子。这种相互作用的分子机制在很大程度上是未知的。我们最近表明，人GAPR-1（hGAPR-1）具有淀粉样特性，导致在酿酒酵母中过表达时形成蛋白质缩合物。在这里，我们表明人类Beclin1（hBeclin1）具有几个预测的淀粉样蛋白生成区域，并且hBeclin1-mCherry在酵母中的过表达也导致荧光蛋白缩合物的形成。令人惊讶的是,hGAPR-1-GFP和hBeclin1-mCherry的共表达导致hBeclin1缩合物的强烈减少。hGAPR-1和Bechlin1表面上已知相互作用位点的突变消除了共表达过程中对缩合物形成的影响，而不会影响单个蛋白质的缩合物形成特性。同样，已知结合hGAPR-1并干扰hGAPR-1和hBeclin1之间的相互作用的hBeclin1衍生的B18肽通过hGAPR-1的共表达消除了hBeclin1缩合物的减少。这些结果表明，相同类型的蛋白质-蛋白质相互作用会干扰hGAPR-1和hBeclin1共表达过程中的缩合物形成，如前所述，它们在高尔基体膜上的相互作用。B18肽的淀粉样特性是，然而,对于与hGAPR-1的相互作用很重要，因为具有降低的淀粉样蛋白生成特性的突变肽也显示出与hGAPR-1的相互作用减少和对hGAPR-1/hBeclin1缩合物形成的干扰减少。我们建议在缩合物形成之前，hGAPR-1和hBeclin1之间发生淀粉样相互作用。
