Biology subjects

Ohsumi, Y.

Publications and source records attributed to Ohsumi, Y..

5 recordsLinked to original sources

bioRxiv2026

Structural insights into interdomain interactions in Entamoeba histolytica APS kinase

The biosynthetic pathway of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a universal and essential metabolic process in many organisms, providing the activated sulfate donor required for the synthesis of diverse sulfated metabolites. However, this pathway has undergone substantial evolutionary diversification among species. In Entamoeba histolytica, PAPS biosynthesis occurs within the mitosomes, mitochondrion-related organelles (MROs), representing a distinctive example of lineage-specific evolutionary adaptation. PAPS synthesis proceeds through a conserved two-step, which is sequentially catalyzed by ATP sulfurylase (AS) and adenosine 5'-phosphosulfate (APS) kinase (APSK). In this study, we focused on E. histolytica APSK (EhAPSK). EhAPSK contains an additional AS-like domain (SLD), although its functional role remains unclear. Here, we determined the crystal structure of full-length EhAPSK at 2.60 [A] resolution and the structure of the truncated EhAPSK lacking APS kinase domain (KD) (EhAPSK{Delta}KD) at 2.10 [A] resolution. Structural analyses revealed that the SLD engages in dynamic contacts with the KD. Furthermore, deletion of the domain and mutational analyses indicated that the SLD significantly influences the catalytic activity of the KD. Based on these findings, we propose a new regulatory mechanism in which transient interdomain interactions modulate APS kinase activity, representing an unique evolutionary adaptation of E. histolytica.

biophysics↗

bioRxiv2025

Structural and mechanistic basis for membrane recognition and activation of the vacuolar lipase Atg15

Summary/Abstract Atg15 is a vacuolar phospholipase B essential for the degradation of intravacuolar vesicles such as autophagic bodies. Despite its central role in cellular membrane turnover, the molecular basis of how Atg15 is activated and selectively acts on internal membranes has remained elusive. Here, by combining all-atom and coarse-grained molecular dynamics (MD) simulations with in vitro and in vivo analyses, we elucidate the structural and mechanistic principles underlying Atg15 activation and substrate recognition. Our simulations revealed that disulfide bonds are critical for maintaining the structural integrity of the catalytic core, while the C-terminal region locks the catalytic center in a closed state that prevents activation. Membrane binding induces a transition to an open state, enabling catalysis. Through MD-guided mutational analysis, we identified three regions crucial for catalytic locking, membrane binding, and substrate recognition, and experimentally confirmed that mutations in these regions inhibit activity. Furthermore, Atg15 preferentially associates with positively curved membranes, providing a potential basis for its selective action on internal vesicular membranes. These findings suggest that Atg15s activity is controlled through multiple regulatory layers to ensure safe and selective membrane degradation.

cell biology↗

bioRxiv2024

A non-canonical CDK, Pho85 regulates the restart of the cell-cycle following stress

Environmental stress induces an arrest of the cell cycle. Thus, release from this arrest is essential for cell survival. The cell-cycle-arrest occurs via the down regulation of the cyclins that drive the main cyclin dependent kinase, CDK1/Cdc28. However, it was not clear how cells escape this potentially fatal arrest. Here we show that prior to the restoration of CDK1/Cdc28 cyclins, a non-canonical CDK, Pho85, initiates a cascade to restart the cell cycle. We demonstrate that following stress, Pho85 phosphorylates the Sch9 kinase, which in turn directly phosphorylates the transcriptional inhibitor Whi5, the yeast analog of RB1/retinoblastoma, and a CDK1 target. This promotes Whi5 translocation from the nucleus, and the release of the stress-induced arrest at G1 phase. In addition, we find that in parallel with Pho85, CDK1/Cdc28 also plays a role in the control of Whi5. Together, these findings provide insights into how cells re-enter the cell cycle during recovery from stress and reveal that a non-canonical CDK and cyclin takes on essential roles and acts via a pathway that functions in parallel with CDK1/Cdc28.

molecular biology↗

bioRxiv2023

The mechanism of Atg15-mediated membrane disruption in autophagy

Autophagy is a lysosomal/vacuolar delivery system that isolates and degrades cytoplasmic material. Following delivery by autophagosomes, cytoplasmic components are released into the vacuole within an autophagic body (AB), which is a single-membrane structure derived from the inner membrane of the autophagosome. This membrane must be disrupted for degradation of the cytoplasmic cargo to occur. The vacuolar proteases Pep4 and Prb1, as well as the lipase Atg15, are known to be necessary for this process, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and use it to show that Atg15 is the sole vacuolar phospholipase and that Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments also reveal that Atg15 is a B-type phospholipase of broad substrate specificity that is likely implicated in the disruption of a range of membranes delivered to the vacuole. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.

biochemistry↗

bioRxiv2022

Isolation and characterization of autophagic bodies from yeast

Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells. In the yeast S. cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation. In this study, we demonstrate a method to purify intact ABs from vacuoles that retain membrane integrity and contain autophagic cargos. This technique offers a valuable tool for the identification of the cargos of autophagy, examination of autophagic cargo selectivity, and biochemical characterization of autophagosome membranes.

molecular biology↗