DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8–9 complex, a paralog of the MCM2–7 replicative helicase. We show that MCM8–9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8–9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8–9 as an alternative replicative helicase.

Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin–proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.

In budding yeast, the presence of glucose, a preferred energy source, suppresses the expression of respiration-related genes through a process known as glucose repression. Conversely, under glucose starvation conditions, Snf1 phosphorylates and activates downstream factors, relieving this repression and allowing cells to adapt. Recently, the Tda1 protein kinase has been implicated in these glucose starvation responses, although its function remains largely uncharacterized. In this study, we demonstrate that Snf1 and yeast glycogen synthase kinase 3-beta (GSK3-β) independently phosphorylate and activate Tda1, which in turn phosphorylates Hxk2 at Ser15. The Ser483 and Thr484 residues of Tda1 are critical for its activation by Snf1, while the Ser509 residue is crucial for its activation by yeast GSK3-β. Importantly, under glucose starvation conditions, the TDA1 deletion mutant shows increased expression of respiration-related genes and a faster growth rate compared to wild-type cells, which is opposite to what is observed in SNF1 and yeast GSK3-β deletion mutants. These findings suggest that Tda1 is activated by Snf1 and yeast GSK3-β, and functions as a suppressor of the glucose starvation signaling.

Saccharomyces cerevisiae proliferates by budding, which includes the formation of a cytoplasmic protrusion called the ‘bud’, into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin–myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

Protein analysis strategies involving targeted protein degradation are powerful approaches to determine gene functions. Auxin-inducible degron (AID) is among the most widely used methods for target protein knockdown. This system enables the rapid depletion of AID-tagged target proteins in an auxin-dependent manner. Various improved AID methods have been developed to date; however, the requirement to tag the target proteins remains a common challenge. Here, we demonstrated the efficiency of an affinity linker-based super-sensitive AID system for the conditional knockdown of target proteins in cultured animal cells and mouse embryos. This system combines the improved AID method with a small protein binder, enabling the control of green fluorescent protein and mCherry fusion proteins. Additionally, this system can be used to degrade tag-free targets, such as Ras proteins. We also developed caged 5-adamantyl-indole-3-acetic acid that precisely controlled targeted protein degradation under light irradiation. This advanced technique aids in the degradation of endogenous proteins of interest and can be used to develop new technologies for localized protein degradation.