Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

Author ContributionsConceptualization: S.N.M., A.J.B and L.M.V.; Investigation, Formal analysis: S.N.M. and D.J.T.; Methodology and Resources: M.M.C., I.L.R., and A.S.; Writing: S.N.M., A.J.B, and L.M.V.; Supervision: M.K., C.P.L., A.J.B, L.M.V. AbstractCellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding, are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show the macromolecular crowding changes less in longer-lived cells in contrast to shorter-lived cells. While the average pH and crowding levels change only modestly with aging, we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively-aged yeast cells.

Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis.