The Plasma Membrane Protein Nce102 Implicated in Eisosome Formation Rescues a Heme Defect in Mitochondria

Abstract

The cellular transport of the cofactor heme and its biosynthetic intermediates such as protoporphyrin IX is a complex and highly coordinated process. To investigate the molecular details of this trafficking pathway, we created a synthetic lesion in the heme biosynthetic pathway by deleting the gene HEM15 encoding the enzyme ferrochelatase in S. cerevisiae and performed a genetic suppressor screen. Cells lacking Hem15 are respiratory-defective because of an inefficient heme delivery to the mitochondria. Thus, the biogenesis of mitochondrial cytochromes is negatively affected. The suppressor screen resulted in the isolation of respiratory-competent colonies containing two distinct missense mutations in Nce102, a protein that localizes to plasma membrane invaginations designated as eisosomes. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15⌬ cells cultured in supplemental hemin. Respiratory function in hem15⌬ cells can also be restored by the presence of a heterologous plasma membrane heme permease (HRG-4), but the mode of suppression mediated by the Nce102 mutant is more efficient. Attenuation of the endocytic pathway through deletion of the gene END3 impaired the Nce102-mediated rescue, suggesting that the Nce102 mutants lead to suppression through the yeast endocytic pathway.Heme is an essential cofactor in eukaryotes and bacteria and functions in a myriad of pathways including oxygen transport, nitric oxide and carbon monoxide sensing, oxygenase reactions, and electron transport reactions (1-3). The heme biosynthetic pathway in eukaryotes, including the yeast Saccharomyces cerevisiae, is a complex process involving eight enzymes (see Fig. 1A) (4). The process starts in the mitochondrial matrix with the condensation of glycine with succinyl-CoA to produce ∂-aminolevulinic acid (ALA) 3 by the enzyme 5-aminolevulinate synthase (Hem1 is the yeast designation) (2, 4). ALA is translocated across the mitochondrial membranes to the cytosol, where the next four enzymes in the pathway convert two ALA molecules to form the intermediate pyrrole porphobilinogen and subsequent formation of coproporphyrinogen III, which is transported back into the mitochondria for conversion to protoporphyrin IX (PPIX). Ferrous iron is inserted into PPIX by ferrochelatase (Hem15), tethered to the inner membrane and facing the matrix, to form the heme cofactor. A defect in any of heme biosynthetic enzymes leads to one of the porphyric disorders in humans, each with a unique phenotype resulting from a buildup of toxic intermediates (3).The mechanism of heme transport between cellular compartments remains poorly understood (4). Nevertheless, significant inroads have been recently made utilizing the heme auxotroph Caenorhabditis elegans, resulting in the identification of heme and porphyrin transporters, including the HRG-1-related heme transporters (4, 5). These transporters have mammalian homologs with similar function and specificity. Additionally, the fel...

The mitochondrial carrier SFXN1 is critical for complex III integrity and cellular metabolism

“…Potentially, there is a difference in the bioavailability of exogenously supplied hemin and endogenously synthesized heme for incorporation into CIII that subsequently affects its stability. Suboptimal delivery of exogenous heme to mitochondria has been previously documented ( Kim et al, 2016 ). This may be a potential explanation for why hemin supplementation may result in only a partial rescue of RC function ( Shetty et al, 2019 ; Vandekeere et al, 2018 ).…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…Pathways for the endocytosis of exogenous heme have been identified in yeast as well, including budding and fission yeast, S. cerevisiase and S. pombe, respectively. In S. cerevisiase, FECH-depleted hem15∆ cells can be rescued by heme in fermentable media but not in non-fermentable media while cells are respiring [204,205]. The mechanism by which yeast cells import exogenous heme and the basis for why mitochondrial heme import is inefficient is not completely clear.…”

“…A wealth of data indicates exogenous heme may be utilized in toto independently of its degradation. In the unicellular model eukaryote, yeast S. cerevisiae, hem1∆ cells, lacking the first enzyme in the heme synthesis pathway, ALA synthase, can be rescued to some extent by heme supplementation [204,205]. Further, in humans, patients suffering from certain porphyrias, a class of disorders characterized by heme synthesis defects, can be treated with intravenous heme and heme-albumin, which rescues heme-dependent enzyme activities in various tissues [214].…”

Mitochondrial Contact Site and Cristae Organizing System (MICOS) Machinery Supports Heme Biosynthesis by Enabling Optimal Performance of Ferrochelatase

“…Hem15-deficient cells exhibit a petite-inducing phenotype that can be rescued genetically through overexpression of ribonucleotide reductase subunit Rnr1, a genetic manipulation known to stabilize mitochondrial DNA in petite mutants (32,50). This finding explains previously described difficulties in establishing a robust genetic complementation of the hem15D mutant with plasmid-borne Hem15 under the control of a strong promoter (29). While the exact molecular underpinnings of the petite-inducing phenotype of hem15D are currently unclear, they might arise from impaired hemylation of mitochondrial matrix hemoproteins such as the yeast flavohemoglobin Yhb1 or translational activator Mss51 (51,52).…”

“…While growth of the hem15 Δ mutant in the presence of glucose can be sustained with either exogenous heme supplementation or re-expression of plasmid-borne Hem15 (7), this is not always the case for respiratory growth (Supplementary Fig. S2A and (29)). Since a fully functional genetic model is critical for further studies, this issue was examined in greater detail.…”