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

细胞的健康和代谢取决于它们利用酶分解膜结合区室（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，尚不清楚。我们认为溶酶体中的酶依赖于Cl-来发挥最佳功能，这是一个基本的、进化保守的过程。因此，提出的研究的主要目的是确定Cl-转运到细胞酸性区室的分子机制。细胞使用超过30-70%的能量来维持跨膜的化学梯度。利用梯度来驱动溶质/代谢物的二次运输，而不需要额外的能量成本。一个典型的例子发生在内吞途径中，其中H+泵送v - atp酶建立并维持H+梯度和溶酶体的酸性pH。然后，H+梯度促进有机溶质通过H+共转运体向外流动和Cl-通过单一交换剂ClC7向内移动。曾有人提出Cl-内流促进酸化，但可能在溶酶体功能中有其他未被充分认识的作用。在我们未发表的研究中，我们发现ClC7仅在成熟的内噬细胞器达到其最大酸性ph后才被纳入其极限膜。我们的研究结果表明，高腔容[Cl-]并没有调节v - atp酶的活性，而是使溶酶体水解酶发挥最佳功能。在我们未发表的研究结果中，我们表明消除ClC7不会改变溶酶体通常的酸性pH值，而是阻止它们降解内吞货物。综上所述，我们未发表的结果表明，酸性内体腔室中腔内Cl-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Cl-交换体的缺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内液的阴离子一起进化。我们推测，内吞途径中的高Cl浓度是由ClCs交换活性驱动的，以支持水解酶的活性。1.具体目标测定（吞噬体）溶酶体[Cl-]和ClC7对建立腔内[Cl-]的贡献。2. 测定体外水解酶对氯的依赖性及体内对氯的影响。3. 研究腔内Cl-在膜传输中的作用，这是室分辨率的基础。的影响。这项工作将有助于解释ClC7突变引起的导致溶酶体储存障碍和生长缺陷的表型。更广泛地说，这些研究将提高我们对跨细胞器膜的H+梯度作为驱动关键溶质（包括Cl-）二次迁移的力的提供者的理解。

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 In our unpublished research, we find that ClC7 is only incorporated into the limiting membrane of mature endocytic organelles after they have reached their maximum acidic pH.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-.