The role of chloride in supporting the degradative capacity of phagosomes and lysosomes

主要目的细胞的健康和新陈代谢依赖于它们使用酶分解膜结合的隔室(如溶酶体)中的大分子的能力。然而，这些隔室的流体环境是如何影响酶活性的，还没有得到很好的描述。我们认为，溶酶体上的酶依赖于氯-来发挥最优功能，这是一个基本的和进化上保守的过程。因此，这项研究的主要目标是确定氯-进入细胞酸性隔间的分子机制(S)。背景与进展细胞使用其30-70%以上的能量来维持跨膜的化学梯度。利用梯度来驱动溶质/代谢物的二次传输，而不需要额外的能量成本。一个最好的例子发生在内吞途径中，H+泵V-ATPase建立并维持溶酶体的H+梯度和酸性pH。然后，H+梯度通过H+-共转运体促进有机溶质的向外流动，并通过单一的交换器ClC7促进氯-的向内移动。CL-内流被认为可以促进酸化，但在溶酶体功能中可能有额外的、未被认识的作用。在我们未发表的研究中，我们发现ClC7只有在成熟的内吞细胞器达到其最大酸性pH时才会被结合到限制膜中。我们的结果表明，高流明的[Cl-]不是调节V-ATPase的活性，而是使溶酶体驻留的水解酶具有最佳的功能。在我们未发表的发现中，我们表明，消除ClC7并不会改变溶酶体通常为酸性的pH，相反，它会阻止它们对内吞货物的降解。综上所述，我们未发表的结果表明，鲁米那氯-在酸性内体隔室的主要功能是促进其水解酶活性。有趣的是，酵母和哺乳动物细胞中氯离子交换器的丢失分别导致生长缺陷和溶酶体储存障碍，这表明细胞器酶可能与腔液中的阴离子一起进化。我们假设，内吞途径中的高氯离子浓度是由CLCs支持水解酶活性的交换活性驱动的。具体目标1.测定(吞噬)溶酶体[Cl-]和ClC7对建立鲁米那[Cl-]的贡献。2.体外测定水解酶对氯离子的依赖性及体内改变氯离子的影响。3.研究鲁米那氯离子在隔室分辨的膜交通中的作用。冲击力。这项工作将有助于解释ClC7突变引起的表型，这些突变导致溶酶体储存障碍和生长缺陷。更广泛地说，这些研究有助于提高我们对跨细胞膜的H+梯度的理解，因为H+梯度是推动关键溶质二次运输的力量的提供者，包括Cl-。

Main Objective The health and metabolism of cells depends on their ability to breakdown large molecules in membrane-bound compartments (e.g. lysosomes) using enzymes. How the fluid environment of these compartments influences enzymatic activity is, however, poorly characterized. We propose that the enzymes resident to lysosomes depend on Cl- to function optimally, a fundamental and evolutionarily conserved process. The main objective of the proposed research is to therefore determine the molecular mechanism(s) underlying the transport of Cl- into the acidic compartments of cells. Summary of background and progress Cells use more than 30-70% of their energy to maintain chemical gradients across membranes. The gradients are exploited to drive the secondary transport of solutes/metabolites without an additional energy cost. A prime example occurs in the endocytic pathway where H+-pumping V-ATPases establish and maintain a H+ gradient and the acidic pH of lysosomes. The H+ gradient then facilitates an outward flux of organic solutes via H+-cotransporters and the inward movement of Cl- via a single exchanger, ClC7. Cl- influx had been proposed to facilitate acidification but may have additional, underappreciated roles in lysosome function. In our unpublished research, we find that ClC7 is only incorporated into the limiting membrane of mature endocytic organelles after they have reached their maximum acidic pH. Rather than regulating the activity of the V-ATPase, our results indicate that high luminal [Cl-] enables the optimal function of lysosome-resident hydrolases. In our unpublished findings, we show that eliminating ClC7 does not alter the normally acidic pH of lysosomes but instead prevents their degradation of endocytosed cargo. Taken together, our unpublished results suggest that the major function of luminal Cl- in acidic endosomal compartments is to facilitate their hydrolase activity. Interestingly, the loss of Cl- exchangers in yeast and mammalian cells leads to growth defects and lysosomal storage disorders respectively, suggesting that organellar enzymes may have evolved alongside with the anions of the luminal fluid. We hypothesize that a high Cl- concentration in the endocytic pathway is driven by the exchange activity of ClCs to support the activity of hydrolytic enzymes. Specific Objectives 1. Measure (phago)lysosomal [Cl-] and the contribution of ClC7 to establishment of luminal [Cl-]. 2. Determine the Cl- dependence of hydrolytic enzymes in vitro and the effect of altering Cl- in vivo. 3. Study the role of luminal Cl- on the membrane traffic that underlies compartment resolution. Impact. This work will help to explain phenotypes arising from ClC7 mutations that lead to lysosomal storage disorders and growth defects. More broadly, these studies stand to improve our understanding of the H+ gradient across organellar membranes as the provider of the force driving the secondary transport of critical solutes, including Cl-.