Biology subjects

Kane, P. M.

Publications and source records attributed to Kane, P. M..

7 recordsLinked to original sources

bioRxiv2025

Early responses to hyperosmotic stress at the yeast vacuole

In yeast, early adaptation to hyperosmotic stress involves organelle-based mechanisms, including synthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) at the vacuole. This low- level signaling lipid drives vacuolar fragmentation and activates the V-ATPase proton pump, which acidifies the vacuole and drives salt sequestration. The vacuole-resident V-ATPase subunit Vph1 interacts with PI(3,5)P2 via its N-terminal domain (Vph1NT), directly linking lipid signaling to proton pump regulation. Under NaCl stress, PI(3,5)P2 rapidly accumulates, triggering increased V-ATPase activity and vacuolar remodeling; these responses are impaired by deficient PI(3,5)P2 synthesis. A Vph1NT-GFP fusion protein with no membrane domain is cytosolic without salt, but upon NaCl addition, rapidly relocalizes to a region adjacent to the vacuole in a PI(3,5)P2- dependent manner. The intensity and duration of this response depend on salt concentration. Vph1NT-GFP returns to the same location upon repeated salt challenge, suggesting that PI(3,5)P2 synthesis occurs at a localized domain/contact site. Disrupting PI(3,5)P2 signaling, V- ATPase activity, or the high osmolarity glycerol pathway, which coordinates long-term transcriptional changes, compromises cellular adaptation to salt, underscoring the integration of lipid signaling and transcriptional regulation in hyperosmotic stress. These findings suggest activation of the V-ATPase, and possibly other targets, by PI(3,5)P2 synthesis provides immediate protection that primes cells for longer-term survival strategies. Significance Statement--Adaptation to high salt involves early responses at organelle membranes and slower transcriptional responses. The vacuolar/lysosomal signaling lipid, PI(3,5)P2 is critical for the early response, but the timing, localization, and targets of salt-induced PI(3,5)P2 synthesis are not fully understood. --Experiments using Vph1NT-GFP as a low-affinity PI(3,5)P2 biosensor suggest lipid synthesis occurs at a specific domain of the vacuolar membrane, with the level and duration of synthesis dependent on salt concentration and V-ATPase activity. A hog1{Delta} mutation ablates the slower response but elevates and extends PI(3,5)P2 activation. --Controlled PI(3,5)P2 synthesis at the vacuole supports V-ATPase-driven salt sequestration; long-term adaptation requires both V-ATPases and the HOG pathway.

cell biology↗

bioRxiv2025

Mdm38/LETM1 couples ion homeostasis and proteostatic mechanisms in the inner mitochondrial membrane

The mitochondrial inner membrane is among the most protein-dense cellular membranes. Its functional integrity is maintained through a concerted action of several conserved mechanisms that are far from clear. Here, using the bakers yeast model, we functionally characterize Mdm38/LETM1, a disease-related protein implicated in mitochondrial translation and ion homeostasis, although the molecular basis of these connections remains elusive. Our findings reveal a novel role for Mdm38 in maintaining protein homeostasis within the inner membrane. Specifically, we demonstrate that Mdm38 is required for mitochondrial iron homeostasis and for signaling iron bioavailability from mitochondria to vacuoles. These processes are linked to the m- AAA quality control protease, whose unrestrained activity disrupts the assembly and stability of respiratory chain complexes in Mdm38-deficient cells. Our study highlights the central role of Mdm38 in mitochondrial biology and reveals how it couples proteostatic mechanisms to ion homeostasis across subcellular compartments.

biochemistry↗

bioRxiv2024

V-ATPase Disassembly at the Yeast Lysosome-Like Vacuole Is aPhenotypic Driver of Lysosome Dysfunction in Replicative Aging

Declines in lysosomal acidification and function with aging are observed in organisms ranging from yeast to humans. V-ATPases play a central role in organelle acidification and V- ATPase activity is regulated by reversible disassembly in many different settings. Using the yeast Saccharomyces cerevisiae as a replicative aging model, we demonstrate that V-ATPases disassemble into their V1 and V0 subcomplexes in aging cells, with release of V1 subunit C (Vma5) from the lysosome-like vacuole into the cytosol. Disassembly is observed after >5 cell divisions and results in overall vacuole alkalinization. Caloric restriction, an established mechanism for reversing many age-related outcomes, prevents V-ATPase disassembly in older cells and preserves vacuolar pH homeostasis. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors, the RAVE complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1{Delta} mutant shortens replicative lifespan; Oxr1 promotes disassembly and an oxr1{Delta} mutation extends lifespan. Importantly, the level of Rav2, a subunit of the RAVE complex, declines in aged cells, and Rav2 overexpression delays V-ATPase disassembly with age. These data indicate that reduced V-ATPase assembly contributes to the loss of lysosome acidification with age, which affects replicative lifespan.

cell biology↗

bioRxiv2024

Structure of yeast RAVE bound to a partial V1 complex

Vacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multi-subunit enzymes consist of a soluble catalytic V1 subcomplex and a membrane-embedded proton-translocating VO subcomplex. The V1 and VO subcomplexes can undergo reversible dissociation to regulate proton pumping, with reassociation of V1 and VO requiring the protein complex known as RAVE (regulator of the ATPase of vacuoles and endosomes). In the yeast Saccharomyces cerevisiae, RAVE consists of subunits Rav1p, Rav2p, and Skp1p. We used electron cryomicroscopy (cryo-EM) to determine a structure of yeast RAVE bound to V1. In the structure, RAVE is a L-shaped complex with Rav2p pointing toward the membrane and Skp1p distant from both the membrane and V1. Only Rav1p interacts with V1, binding to a region of subunit A not found in the corresponding ATP synthase subunit. When bound to RAVE, V1 is in a rotational state suitable for binding the free VO complex, but it is partially disrupted in the structure, missing five of its 16 subunits. Other than these missing subunits and the conformation of the inhibitory subunit H, the V1 complex with RAVE appears poised for reassembly with VO.

biochemistry↗

bioRxiv2023

Human V-ATPase a-subunit isoforms bind specifically to distinct phosphoinositide phospholipids

V-ATPases are highly conserved multi-subunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue and organelle specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms. We hypothesize that the aNT domains of the human isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. Bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP binding sites, potential binding sites in the HuaNT domains were identified by sequence comparisons and existing subunit structures and models. Mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data also suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.

biochemistry↗

bioRxiv2022

Chimeric a-subunit isoforms generate functional yeast V-ATPases with altered regulatory properties in vitro and in vivo

V-ATPases are highly regulated, multi-subunit proton pumps that acidify organelles. The V-ATPase a-subunit is a two-domain protein containing a C-terminal transmembrane domain that participates in proton transport and a N-terminal cytosolic domain (aNT) that acts as a regulatory hub, integrating environmental inputs to regulate assembly, localization, and V-ATPase activity. Tissue- and organelle-specific a-subunit isoforms exist in most organisms, but how regulatory inputs are decoded by aNT isoforms is unknown. The yeast S. cerevisiae encodes only two organelle-specific a-isoforms, Stv1 in the Golgi and Vph1 in the vacuole. Based on recent structures, we designed chimeric yeast aNTs in which the globular proximal and distal ends are exchanged. The Vph1 proximal-Stv1 distal (VPSD) aNT chimera binds to the glucose-responsive RAVE assembly factor in vitro but exhibits little binding to phosphoinositide lipids that activate V-ATPases. The Stv1 proximal-Vph1 distal (SPVD) aNT lacks RAVE binding but binds more tightly to phosphoinositides than Vph1 or Stv1. When attached to the Vph1 C-terminal domain in vivo, both chimeras complement growth defects of a vph1{Delta} mutant, but only the SPVD chimera exhibits wild-type V-ATPase activity. Cells containing the SPVD chimera adapt more slowly to a poor carbon source than wild-type cells but grow more rapidly than wild-type after a shift to alkaline pH. This is the first example of a "redesigned" V-ATPase with altered regulatory properties and adaptation to specific stresses.

biochemistry↗

bioRxiv2021

Defining the Yeast Resistome through in vitro Evolution and Whole Genome Sequencing

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe, Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6 transcription factors, YRR1 and YRM1 (p < 1x 10 -100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution.

genetics↗