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 hem15Δ cells cultured in supplemental hemin. Respiratory function in hem15Δ 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.
Highlights
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 [1,2,3]
Observed when the gene encoding for 5-aminolevulinate synthase (HEM1) (Fig. 1C) or uroporphyrinogen decarboxylase (HEM12) was deleted, indicating that the inability to respire in these cells is a result of the lesion in the heme biosynthetic pathway, as opposed to a loss of the specific proteins
The Respiratory Defect in hem15⌬ Cells Is Suppressed by Mutations in the Plasma Membrane Protein Nce102—In an attempt to gain insights into the inefficient transport of heme to mitochondria to support formation of respiratory complexes, we screened for extragenic compensatory suppressors by plating hem15⌬ cells at high cell density on glycerol/lactate-containing medium supplemented with 20 M exogenous hemin
Summary
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 [1,2,3]. The presence of the Nce102 mutant alleles enabled formation of the mitochondrial respiratory complexes and respiratory growth in hem15⌬ cells cultured in supplemental hemin.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
