Exploring the protein interactions and phosphorylation status of Maf1 during optimal growth and stress conditions in the human parasite Leishmania major

Maf1 was originally described as a repressor of RNA polymerase III transcription in response to stress conditions in yeast, but recent work has shown that it is involved in several other functions across eukaryotes. Here we characterize Maf1 in Leishmania major (LmMaf1), a protozoan parasite that exhibits unconventional mechanisms of gene expression. Despite sequence divergence, LmMaf1 contains the conserved domains A, B and C. Four phosphorylated residues were detected in domains B and C in mid-logarithmic phase parasites by mass spectrometry, an unexpected result since phosphorylation has not been reported in these domains in other organisms. Notably, no phosphorylated amino acids were found in stationary phase parasites, suggesting that nutrient stress might induce dephosphorylation of LmMaf1. Indirect immunofluorescence assays with a cell line that expresses LmMaf1 fused to a PTP tag showed that Maf1 mainly localizes to the nucleus. Interestingly, unlike other species, the LmMaf1 subcellular localization did not change in stressed and stationary-phase parasites. Chromatin immunoprecipitations revealed the association of LmMaf1 with RNAP III-dependent genes and other genomic regions. Tandem affinity purifications allowed us to identify the proteins that interact with LmMaf1 under optimal growth and heat stress. Although similar groups of proteins were identified in both normal and heat-stressed cells, including RNA polymerase subunits, the relative abundance of several of them showed significant changes. Also, multiple proteins were identified exclusively under normal growth or heat stress conditions. Altogether, these results suggest the involvement of Maf1 in diverse roles that help to maintain general cellular homeostasis both under normal and stressful conditions in L. major, including transcription control, post-transcriptional regulation of mRNA expression, lipogenesis, and ribosomal biogenesis.