Loading...
Endolysosomal phosphoinositides PI3P and PI(3,5)P2 regulate mammalian lysosomal V-ATPases containing the V0 a1-isoform
Journal Title
Readers/Advisors
Kane, Patricia
Journal Title
Term and Year
Publication Date
2025-06
Book Title
Publication Volume
Publication Issue
Publication Begin
Publication End
Number of pages
Files
Research Projects
Organizational Units
Journal Issue
Abstract
Lysosomal acidification is critical for maintaining luminal acidic pH─ requirement for lysosome function in macromolecule processing, metabolite transport, and trafficking. Conversely, impaired acidification of lysosomes is associated with a wide array of diseases, including lysosomal storage disorders (LSDs) and multiple neurological disorders. The multi-subunit vacuolar-ATPase (V-ATPase) enzyme is the key player in acidifying lysosomes. V-ATPase, an ATP-dependent proton translocating pump, causes the lumenal acidification of lysosomes by proton translocation across the lysosomal membrane, emphasizing the importance of regulating V-ATPase function in lysosomes. The regulation of V-ATPase proton pump is multifaceted. The multi-subunit V-ATPase has a membrane-embedded V0 sub-complex, where the V0 a-subunit dictates organelle-specific distribution and assembly of V-ATPases. The V0 a-subunit has four isoforms (a1-a4) in mammals, whereas the a1 and a3 isoforms both localize to endolysosomal compartments and are involved in lysosomal acidification in many cells. However, the a-isoform-specific regulation of lysosomal V-ATPase function is not completely understood. Here, we used 4T1 mouse breast cancer cell line and it's a1-isoform knockout and partial a3-knockdown counterparts to unveil a-isoform-specific regulation and function of V-ATPases in lysosomes. Pulse-chase endocytosis of FITC-Dextran revealed that V-ATPase-mediated lysosomal acidification still occurs both in a1KO and a3KO cell lines as well as wild-type (WT) 4T1 cells. Regardless of the complete loss of a1 or partial expression of a3; the lysosomal pH, luminal proteolytic activity remain unperturbed in a1KO and a3KD cells. Partial overlapping of V0a1 and V0a3 antigen under immunofluorescence microscopy corroborates the redundant functions of both isoforms and suggests the rescue of lysosomal pH and function by the alternative a-isoform in case any one a-isoform is lost or partially expressed. Interestingly, lysosome-containing cell lysates from 4T1-a1KO and 4T1-a3KD cell lines fail to re-establish the pH gradient in vitro, indicating a possibility of different regulatory mechanisms for a1- and a3-containing lysosomes. Removal of PI3P and PI(3,5)P2 from WT and mutant cells unveiled that endolysosomal PIP removal reduces only the a1-containing V-ATPase proton pumping activity. We previously reported that the N-terminal cytosolic domain (NT) of endolysosomal V0 a1 binds specifically to endolysosomal phosphoinositides (PIPs) ─ PI3P and PI(3,5)P2. A potential PIP binding site was identified in a1 isoform. Mutation in this site abolishes the interaction with these PIPs in vitro. Given this finding, we exploited a CRISPR-Cas9-based homology-directed repair approach (HDR) to introduce this specific mutation into genomic V0 a1 of 4T1 cells. We noticed no notable changes in lysosomal acidification, pH and cathepsin B protease activity in the a1-PIP binding mutant. However, due to the Y214VH>AVA mutation in a1NT, the resulting a1-PIP mutant displayed significantly reduced proton pumping activity, a similar pattern noted in the untreated a1KO cell lysate. Endolysosomal PIPs removal and generation of a1-PIP binding mutant cells confirmed the endolysosomal PIP-dependent regulation of a1- containing V-ATPase activity in lysosomes, where PI3P and PI(3,5)P2 interacts with the 'Y214VH' residue of a1NT. Altogether, the findings of our study elucidates an unresolved mechanisms of V-ATPase regulation and provide new insight for target-based development of therapeutics to treat lysosomal disorders caused by V-ATPase dysregulation and dysfunction.
Citation
DOI
Description
Accessibility Statement
If this SOAR repository item is not accessible to you (e.g. able to be used in the context of a disability), please email libsuppt@upstate.edu.