Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these polyphosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmania in vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4^–) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. major VTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCF^Grr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second SCF^Grr1 responsive degron in Med13. Deletion of Snf1 resulted in nuclear retention of cyclin C and failure to induce mitochondrial fragmentation. This degron was able to confer oxidative-stress-induced destruction when fused to a heterologous protein in a Snf1 dependent manner. Although snf1∆ mutants failed to destroy Med13, deleting the degron did not prevent destruction. These results indicate that the control of Med13 degradation following H₂O₂ stress is complex, being controlled simultaneously by CWI and MAPK pathways.

The cell cycle is the process by which a cell duplicates its DNA during S-phase and divides its chromosomes during M-phase, creating two genetically identical daughter cells. Cell cycle events are ordered by synthesis and degradation of key cell regulators and by phosphorylation and dephosphorylation of numerous substrates. Phosphorylation can alter the activity, interactions or subcellular localization of a protein. A substrate’s phosphorylation status is the readout of competing activities of kinases and phosphatases that target each of its phosphorylation sites. In our recent study (EMBO J. 37, e98745), we performed time-resolved global phosphoproteome analysis of a period during the cell cycle known as mitotic exit. During this time, numerous cell biological events happen in fast succession but in strict order. First, at the metaphase to anaphase transition, the mitotic spindle elongates to pull maximally condensed chromosomes to opposite cell halves. Shortly after that, spindles disassemble and chromosomes decondense, before finally cell division is completed by cytokinesis. Our time-resolved phosphoproteome analysis of this period in budding yeast provided a survey of the principles of phosphoregulation used to order these events.

The gammaretroviral gag cleavage product, p12, is essential for replication at both early and late stages of the virus life cycle. During the early stage of infection, the viral core is released into the cytoplasm, the viral RNA genome is reversed transcribed to cDNA and this viral DNA is then integrated into the host cell chromatin to form a provirus. The p12 protein has N- and C-terminal domains (NTD and CTD) that are required for steps leading up to integration, but the molecular details of their functions remain poorly characterised. Using the prototypic gammaretrovirus, murine leukemia virus (MLV) as a model, we recently showed that the NTD of p12 directly binds to and stabilises the capsid (CA) lattice of the viral core. Alterations to the CTD of MLV p12 prevented the viral pre-integration complex (PIC) tethering to host chromatin in mitosis, and this could be partially rescued by addition of a heterologous chromatin binding motif. In this study we demonstrated that the CTD of p12 directly binds to nucleosomal histone proteins, targeting not only p12 but also CA to mitotic chromatin. Additionally, cell-cycle-dependent phosphorylation of p12 appeared to increase the affinity of p12 for chromatin in mitosis relative to interphase. Thus, we have revealed how p12 can link the CA-containing PIC to mitotic chromatin, ready for integration. Importantly, we observed that direct binding to nucleosomes is a conserved feature of p12 orthologs across the gammaretrovirus genus and that the nucleosomal docking site is potentially shared with that of spumaretroviral Gag proteins.