Author ORCID Identifier

0000-0001-9008-3134

Date of Graduation

12-2020

Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Michael X. Zhu, Ph.D

Committee Member

Guangwei Du, Ph.D

Committee Member

Ilya Levental, Ph.D

Committee Member

Kartik Venkatachalam, Ph.D

Committee Member

Sheng Zhang, Ph.D

Abstract

Two Pore Channels (TPCs) are endolysosomal ion channels that are permeable to sodium and calcium. Defects in TPCs have been implicated to impair vesicle trafficking, autophagy and cell metabolism control; however, the detailed mechanism remains largely unknown. In this study, I show that TPCs are critical for appropriate cargo delivery to the lysosomes and deletion of either TPC1 or TPC2 leads to delayed clearance of autophagosomes, resulting in enlarged lysosomes and accumulated contents inside the lysosomes. Cells with both TPC deleted also exhibit 50% reduction in lysosomal amino acids under normal culture conditions, leading to reduced homeostatic mTORC1 activation.

Glutamine is one of the most abundant amino acid in the cells. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which is the precursor of multiple metabolites, including -ketoglutarate and glutathione. Here, I show that ammonia generated during GLS1/2 mediated glutaminolysis regulates lysosomal pH in a dose-dependent manner. In mouse embryonic fibroblast (MEF) cells, as well as immortal cell lines, including L929 and HeLa cells, deprivation of total amino acids for 1 hour increased lysosomal degradation capacity as shown by the enhanced turnover of LC3-II and endocytosed DQ-BSA. Removal of glutamine, but not any other amino acids, from the medium recapitulated the enhancement of lysosomal degradation. This lysosomal degradation boost is likely due to the increased lysosomal acidification, as lysosomal pH in MEF and L929 cells is at least ~0.5 unit lower in glutamine-free medium than in complete medium. Addition of glutamine to glutamine-deprived L929 cells elevated lysosomal pH from 4.7 to 5.5 within 90 seconds, which was dramatically attenuated by inhibiting GLS1 or using ammonia scavengers. In MEF cells, mTORC1 activity did not drop immediately in response to amino acid starvation; however, this delay period was markedly shortened by supplementing the amino acid-starved cells with glutamine or treatment with NH4Cl, revealing the importance of accelerated lysosomal degradation in the sustention of mTORC1 activity immediately following starvation.

Together, my study demonstrated Na+ and NH3 as regulators of lysosomal ionic and metabolomic homeostasis. These findings unravel TPCs and glutamine as novel regulators of mTORC1 and lysosomal degradation function.

Keywords

Lysosomal Function, Ca2+ signaling, Two Pore Channels, mTORC1, Glutamine Metabolism, Autophagy, Rapid Organelle Purification, Lysosome Ion/Metabolite Homeostasis

Available for download on Saturday, December 18, 2021

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