Biology subjects

Garcia-Guerrero, A. E.

Publications and source records attributed to Garcia-Guerrero, A. E..

4 recordsLinked to original sources

bioRxiv2024

Insights into the Translational Activation Mechanisms of the COX1 mRNA in Yeast Mitochondria

Mitochondrial translation is a critical regulatory step in mitochondrial genome expression. In Saccharomyces cerevisiae, translational activators are believed to bind to the 5 UTRs of their target mRNAs to position the mitochondrial ribosome at the start codon. Pet309 and Mss51 are translational activators of COX1 mRNA, which encodes subunit one of cytochrome c oxidase. Pet309 physically interacts with COX1 mRNA, but no direct interaction of Mss51 with its target mRNA has been detected. Currently, the mechanisms underlying translational activation of COX1, or any other mitochondrial gene, remain poorly understood. To explore in depth the mechanism of COX1 mRNA translational activation, we studied the association of Pet309 and Mss51 with the mitochondrial ribosome. Both Pet309 and Mss51 interact with the mitoribosome regardless of the presence of COX1 mRNA or of each other. Pet309s association with the ribosome and with COX1 mRNA depends on its N-terminal domain. These findings indicate that Pet309 and Mss51 stably interact with the mitoribosome independently of an active translation. By integrating our data with previously published research, we propose a new mechanism of COX1 mRNA translation activation. SUMMARY STATEMENTYeast mitochondrial mRNAs require translational activators by an almost unknown mechanism. Based on our findings on Mss51 and Pet309 function, we present a new model for translation of the COX1 mRNA

molecular biology↗

bioRxiv2024

Biogenesis of cytochromes c and c1 in the electron transport chain of malaria parasites

Plasmodium malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and a key antimalarial drug target. ETC function requires cytochromes c and c1 that are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate biogenesis of the mature cytochrome c or c1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, Plasmodium parasites are like yeast and encode two separate HCCS homologs thought to be specific for heme attachment to cyt c (HCCS) or cyt c1 (HCC1S). To test the function and specificity of P. falciparum HCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c1 biogenesis and caused lethal ETC dysfunction that was not reversed by over-expression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in E. coli of cyt c hemylation by P. falciparum HCCS and HCC1S strongly suggest that both homologs are essential for mitochondrial ETC function and have distinct specificities for biogenesis of cyt c and c1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

microbiology↗

bioRxiv2023

Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

microbiology↗

bioRxiv2022

The cytochrome b carboxyl-terminal region is necessary for mitochondrial Complex III assembly

Mitochondrial bc1 complex from yeast has ten subunits, but only Cytochrome b (Cytb) subunit is encoded in the mitochondrial genome. Cytb has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cytb synthesis, and together with Cbp4 induce Cytb hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytb synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Since Qcr7 resides near the Cytb carboxyl-region, we wondered whether this region is important for Cytb synthesis/assembly. Although deletion of the Cytb C-region did not abrogate Cytb synthesis, the assembly-feedback regulation was lost, so Cytb synthesis was normal even if Qcr7 was missing. Mutants lacking the Cytb C-terminus were non-respiratory due to absence of fully assembled bc1 complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work we demonstrate that the C-terminal region of Cytb is critical for regulation of Cytb synthesis and bc1 complex assembly.

biochemistry↗