A Peptide from Budding Yeast GAPDH Serves as a Promising Antifungal against Cryptococcus neoformans

Cryptococcus neoformans and Cryptococcus gattii can cause cryptococcosis, which has a high mortality rate. To treat the disease, amphotericin B and fluconazole are often used in clinic.

Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast

Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells became tolerant to severe environmental stresses. Intracellular RNA concentration decreased, and massive downregulation of ribosomal biosynthesis genes occurred. We proved that the expression of new proteins during purine starvation is critical for cells to attain stress tolerance phenotype Msn2/4p targets are upregulated in purine-starved cells when compared to cells cultivated in purine-rich media. The overall transcriptomic response to purine starvation resembles that of stationary phase cells. Our results demonstrate that the induction of a strong stress resistance phenotype in budding yeast can be caused not only by natural starvation, but also starvation for metabolic intermediates, such as purines.

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

A peptide derived from GAPDH enhances resistance to DNA damage in budding yeast

Social behaviors do not only exist in higher organisms but are also present in microbes that interact for the common good. Here, we report that budding yeast cells interact with their neighboring cells after exposure to DNA damage. Yeast cells irradiated with DNA-damaging ultraviolet light secrete signal peptides that can increase the survival of yeast cells exposed to DNA-damaging stress. The secreted peptide is derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and it induced cell death of a fraction of yeast cells in the group. The data suggest that the GAPDH-derived peptide serves in budding yeast’s social interaction in response to DNA-damaging stress. Importance Many studies have shown that microorganisms, including bacteria and yeast, display increased tolerance to stress after exposure to the same stressor. However, the mechanism remains unknown. In this manuscript, we report a striking finding that S. cerevisiae cells respond to DNA damage by secreting a peptide that facilitates resistance to DNA-damaging stress. Although it has been shown that GAPDH possesses many key functions in cells aside from its well-established role in glycolysis, this study demonstrated that GAPDH is also involved in the social behaviors response to DNA-damaging stress. The study opens the gate to an interesting research field about microbial social activity for adaptation to a harsh environment.

Structure of a TRAPPII-Rab11 activation intermediate reveals GTPase substrate selection mechanisms

Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The TRAPP complexes activate these GTPases via nucleotide exchange using a shared set of core subunits. The basal specificity of the TRAPP core is towards Rab1, yet the TRAPPII complex is specific for Rab11. A steric gating mechanism has been proposed to explain TRAPPII counterselection against Rab1. Here we present cryoEM structures of the 22-subunit TRAPPII complex from budding yeast, including a TRAPPII-Rab11 nucleotide exchange intermediate. The Trs130 subunit provides a ″leg″ that positions the active site distal to the membrane surface, and this leg is required for steric gating. The related TRAPPIII complex is unable to activate Rab11 due to a repulsive interaction, which TRAPPII surmounts using the Trs120 subunit as a ″lid″ to enclose the active site. TRAPPII also adopts an open conformation enabling Rab11 to access and exit from the active site chamber.