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Abstract: Mitochondria are important organelles in most eukaryotic organisms as they supply the majority of the ATP needed for cellular functions. Although mitochondria contain their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus. Thus, when mitochondria experience stress they signal the nucleus to alter gene expression and provide a response to the stress. In Neurospora crassa, one model for studying mitochondrial signaling is the induction of alternative oxidase (AOX). This enzyme is a mitochondrial protein encoded by the nuclear aod-1 gene and serves as an alternative terminal oxidase in the electron transport chain. Under normal growth conditions, AOX is not expressed. However, induction of the AOX occurs when cells are grown in the presence of drugs that decrease the function of the standard electron transport chain or when they carry mutations affecting the standard electron transport chain. The induction of AOX requires the function of two Zn(II)Cys6 transcription factors named AOD2 and AOD5. In this study, I demonstrated that both AOD2 and AOD5 localize to the nucleus regardless of whether or not cells are grown under conditions that induce AOX. AOD2 and AOD5 were also shown to exist as a heterodimer in vivo. The orthologues of AOD2 and AOD5 in other fungi have been shown to regulate the expression of genes required for gluconeogenesis. Here, I have shown that the growth of N. crassa on poor carbon sources was severely impaired in the absence of AOD2 or AOD5. Moreover, the expression of the gene encoding phosphoenolpyruvate carboxylase (PEPCK), which is required for gluconeogenesis, was greatly reduced in the absence of AOD2. However, no AOX was detected in cells grown in poor carbon sources, suggesting that the action of AOD2 and AOD5 in regulating gluconeogenesis and AOX is complex and might occur via response to different signals. To investigate whether AOD2 and AOD5 are involved in other cellular functions, I performed chromatin immunoprecipitations followed by next generation sequencing (ChIP-seq) to investigate the global binding of AOD2 and AOD5 in cells grown in the presence and absence of chloramphenicol, a drug that inhibits mitochondrial protein synthesis and, thus, indirectly affects the function of the standard electron transport chain. I focused on the 65 ChIP-seq peaks that exhibited more than four-fold enrichment from strains expressing tagged versions of AOD2 and AOD5 that could be immunoprecipitated with commercial antibodies as compared to a wild-type control strain with no tagged proteins. ChIP-seq was done following growth in both AOX inducing and non-inducing conditions. I found that the 5’ regions of aod-1 and PEPCK are constitutively bound by AOD2 and AOD5. Furthermore, AOD2 and AOD5 also bind to the 5’ regions of many other genes. Most of the binding sites for the 65 peaks contain at least one AOX induction motif, which was previously shown to bind AOD2 and AOD5 using in vitro assays. Several of the genes associated with the 65 peaks were validated as requiring AOD2 and AOD5 for normal expression using RT-qPCR. This study suggests that not only do AOD2 and AOD5 regulate the AOX induction pathway and gluconeogenic pathway, but they also appear to play larger roles in energy homeostasis and reprogramming of metabolism under oxidative stress.

Abstract: The TOB (Topogenesis of Outer membrane β-barrel proteins) complex is composed of the core proteins Tob55 (Sam50), Tob37 (Sam37) and Tob38 (Sam35). The complex facilitates the insertion of all β-barrel and some α-helically anchored integral proteins into the mitochondrial outer membrane (MOM). Little is understood about the architecture of the TOB complex and the protein-protein interactions required for complex stability and function. We have shown that the three TOBcore proteins are essential in Neurospora crassa making in vivo analysis of simple TOB knockout strains impossible. However, I have shown that severe depletion of Tob37 or Tob38 protein in heterokaryotic cultures leads to disruption of β-barrel protein import as well as the import of at least one α-helically anchored MOM protein, Tom22. In addition, I identified a key topological feature located near the C-terminus of Tob37. This region contains novel dual α-helices, one of which acts as the transmembrane domain (TMD) anchor for Tob37 that stabilizes the association of the protein with the complex. I have also shown that Tob38 is severely reduced in the absence of Tob37 and that Tob38 likely interacts with Tob37. In a second project I examined the topology of N. crassa Tom40, the essential core β-barrel protein of the TOM (Translocase of the Outer Membrane) complex. Tom40 forms the pore through which the vast majority of mitochondrial proteins must pass through to enter the organelle. I used substituted cysteine accessibility mapping (SCAM) to define the topology and identify the structural β-strand arrangement of specific amino acid residues in a region of Tom40. These data, together with previous data from our laboratory allowed the development of a partial model based on empirical molecular evidence that we compared to various in silico developed models. The analysis favors the recently proposed model of a β-barrel protein containing 19 β-strands that was developed in other laboratories using the structure of the related protein VDAC (or porin) as a model.

Abstract: Alternative oxidase (AOX) is a single protein enzyme that carries electrons from ubiquinol to molecular oxygen. AOX is induced in many organisms, including Neurospora crassa, when the standard electron transport chain is blocked through various inhibitors or mutations. A knockout (KO) for a dual specificity phosphatase, termed YVH1, cannot properly induce AOX and has a slow growth phenotype. YVH1 contains a HCX5R phosphatase domain and a zinc-binding domain. YVH1 localizes to the nucleus and cytosol equally under all conditions studied. The phenotype of the KO is rescued with constructs that contain only a functional zinc-binding domain. The phosphatase domain appears to be non-essential. The protein has been shown to play a role in ribosome biogenesis in yeast. Preliminary RNA-seq experiments revealed that expression of 17% of the 10,000 protein coding genes of N. crassa is altered at least 2 fold by loss of yvh1 when grown in inducing conditions.