Mitochondria Directly Acidify Lysosomes Through Physical Contact Sites

Lysosomes are critical organelles that maintain cellular health by degrading waste proteins, recycling nutrients, and supporting autophagy — the cellular self-cleaning process. Their function depends entirely on maintaining a strongly acidic internal pH (as low as 4.5). The textbook model holds that lysosomes acidify by importing free protons from the neutral cytosol via the V-ATPase proton pump. However, this paper by Liu et al. at the Buck Institute challenges that model with a surprising discovery: mitochondria are a primary proton source for lysosomes, and they deliver those protons through direct membrane contact sites — not through the bulk cytoplasm.

The problem with the classical model is a numbers game. A single yeast cell contains fewer than 3,000 free cytosolic protons, while cytosolic buffering molecules (phosphate, proteins) are 5–10 orders of magnitude more abundant and compete aggressively for those protons. This means the V-ATPase is constantly outcompeted before it can capture enough protons to acidify the lysosome. The researchers hypothesized that membrane contact sites between mitochondria and lysosomes/vacuoles (Mito-Lyso/Vac contacts) create a privileged microenvironment where protons pumped out of mitochondria by the electron transport chain (ETC) are shielded from cytosolic competitors and transferred directly to the V-ATPase.

Using yeast as a primary model, the team showed that overexpression of Tom70 — which preserves mitochondrial membrane potential during aging — prevented age-associated vacuole de-acidification. Blocking ETC complex III with antimycin A significantly impaired vacuolar acidification, while inhibiting the mitochondrial F1-F0 ATPase with oligomycin had no effect, confirming that the proton gradient itself (not ATP production) was the key variable. Overexpressing Vam6 or Ypt7, the yeast proteins that tether mitochondria to vacuoles (the vCLAMP complex), enhanced vacuolar acidification, while deleting them impaired it. To eliminate indirect effects of these multifunctional proteins, the team engineered a synthetic Mito-Vac linker using an mCherry-tagged outer mitochondrial membrane protein and a matching nanobody anchored to the vacuole membrane. This artificial tether increased Mito-Vac contacts and significantly enhanced vacuolar acidification and autophagic flux, effects specific to the mitochondria-vacuole connection (an ER-vacuole linker had no effect).

Molecular simulations of proton diffusion in a cytoplasm densely packed with proteins and phosphate confirmed that mitochondrial protons are rapidly neutralized by cytosolic competitors unless the vacuole membrane is in immediate proximity. In vitro experiments using isolated organelles suspended in pH 7.5 buffer — removing all cytosolic contributions — demonstrated that vacuoles physically attached to mitochondria via the synthetic linker became significantly more acidic in the presence of NADH and an ATP regeneration system. Removing NADH or adding antimycin A abolished this acidification, directly proving that ETC-driven proton pumping at the contact site acidifies the vacuole.

In aging yeast mother cells, Mito-Vac contacts progressively decline with replicative age, correlating with vacuolar de-acidification. Strikingly, daughter cells — which undergo rejuvenation and reset their aging clock — inherit the de-acidified vacuoles from old mothers but rapidly re-acidify them, a process that depends on the restoration of Mito-Vac contacts in daughter cells. In human senescent cells (induced by ionizing radiation or replicative exhaustion), mitochondria-lysosome contacts similarly declined and lysosomes became less acidic. Expressing the synthetic Mito-Lyso linker in senescent human cells restored lysosomal acidification, rescued autophagic flux, and significantly reduced the secretion of major SASP (senescence-associated secretory phenotype) inflammatory factors including IL-6 and IL-8, establishing a direct mechanistic link between mitochondria-lysosome coupling, lysosomal function, and the inflammatory hallmarks of cellular senescence.