The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex creates contact sites between the endoplasmic reticulum and mitochondria, playing crucial roles in interorganelle communication, mitochondrial fission, mtDNA inheritance, lipid transfer, and autophagy. The mechanism regulating the number of ERMES foci within the cell remains unclear. Here, we demonstrate that the mitochondrial membrane protein Emr1 contributes to regulating the number of ERMES foci. We show that the absence of Emr1 significantly decreases the number of ERMES foci. Moreover, we find that Emr1 interacts with the ERMES core component Mdm12 and colocalizes with Mdm12 on mitochondria. Similar to ERMES mutant cells, cells lacking Emr1 display defective mitochondrial morphology and impaired mitochondrial segregation, which can be rescued by an artificial tether capable of linking the endoplasmic reticulum and mitochondria. We further demonstrate that the cytoplasmic region of Emr1 is required for regulating the number of ERMES foci. This work thus reveals a crucial regulatory protein necessary for ERMES functions and provides mechanistic insights into understanding the dynamic regulation of endoplasmic reticulum-mitochondria communication.

In recent decades, tremendous progress has been made in understanding serine/threonine protein phosphatase-dependent pathways, which are involved in the regulation of numerous processes, including secretion, cell motility, cell cycle, gene transcription, and cell metabolism (1-7). Serine/threonine phosphorylation, which is regulated by many kinases, can be reversed by a few phosphatases that are targeted to substrates via dozens of regulatory subunits (7,8). Currently, serine/threonine protein phosphatases (PPs) are grouped into two structurally distinct families: the PPP family (PP1, PP2A, and PP2B) and the PPM family (PP2C and pyruvate dehydrogenase phosphatase) (9). The PP2A heterotrimeric protein complex, which consists of a cascade of three subunits, namely, a catalytic subunit (C) and a structural subunit (A) associated with a third, hypothetically competitive and variable regulatory subunit (B), represents a highly conserved eukaryotic signal transduction system that is present in many organisms, from yeasts to humans. The activity of PP2A, along with its subcellular localization, is determined by B-family subunits (10-14). In mammals, PP2A is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism (15). Therefore, PP2A is one of a few serine/threonine-specific phosphatases in the cell, and its complex structure and regulation system guarantee its various functions. The ability of this widely distributed heterotrimeric enzyme to function on a diverse array of substrates is largely controlled by the nature of its regulatory Bfamily subunits. Thus, multiple isoforms of the B-family regulatory subunit have been isolated from different organisms and grouped into three classes (B, B=, and BЉ) based on their structural similarities (16,17). The structural variations between these families support the hypothesis that the B-family regulatory subunit controls enzyme activity and specificity, such that different activities of PP2A and subcellular localization are determined by Bfamily regulatory subunits. However, one challenge we face is the potential for redundancy in the regulatory subunits' function of assigning PP2A function in mammals (18)(19)(20). Therefore, it is difficult to approach the function of PP2A globally by knocking out one of the regulatory subunits. In contrast, simple eukaryotic organisms, such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast), which contain only a few isoforms of B-family regulatory subunits, are excellent model systems for studying how B-family regulatory subunits function in regulating PP2A activity. In fission yeast, there is only one B subunit of PP2A-Pab1 and two of the B=-encoding genes (par1 and par2) (21-24). Moreover, mutational studies in fission yeast have demonstrated that B regulatory subunits play crucial roles during the processes of cytokinesis, cell morphogenesis, and cell wall synthesis (25,26). Similarly, in budding yeast, studies have shown that the B class has only one ...

SummaryTimely cytokinesis/septation is essential for hyphal growth and conidiation in Aspergillus nidulans. Genetic analyses have identified that A. nidulans has components of the septum initiation network (SIN) pathway; one of these, SEPH, is a key player for early events during cytokinesis. However, little is known about how the SEPH kinase cascade is regulated by other components. Here, we demonstrate that the phosphoribosyl pyrophosphate synthetase family acts antagonistically against the SIN so that the downregulation of AnPRS family can bypass the requirements of the SIN for septum formation and conidiation. The transcription defect of the Anprs gene family accompanied with the reduction of AnPRS activity causes the formation of hyper-septation as well as the restoration of septation and conidiation in the absence of SEPH. Clearly, the timing and positioning of septation is related to AnPRS activity. Moreover, with the extensive yeast two-hybrid analysis and rescue combination experiments, it demonstrated that AnPRS members are able to form the heterodimers for functional interacting entities but they appear to contribute so unequally that Anprs1 mutant display relatively normal septation, but Anprs2 deletion is lethal. Thus, compared to in yeast, the AnPRS family may have a unique regulation mechanism during septation in filamentous fungi.

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus. Despite the essentiality of AfERG10B having been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus. The reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved, revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed the antifungal drug development pipeline. IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus. Current therapeutic options rely on a limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria and is essential for A. fumigatus survival and morphological development. In combination with structural studies, we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.