The heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of mitochondrial dynamics and ER contact sites, regulate heme mobilization to the nucleus

Abstract

Heme is an iron-containing cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell.However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. We found that heme distribution occurs simultaneously via parallel pathways. In fact, surprisingly, we find that trafficking to the nucleus is ~25% faster than to the cytosol or mitochondrial matrix. Moreover, we discovered that the heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Altogether, our results indicate that the nucleus acquires heme faster than the cytosol or mitochondrial matrix, presumably for mitochondrial-nuclear retrograde signaling, and that GTPases that regulate mitochondrial dynamics and ER contact sites are hard-wired to cellular heme distribution systems. nuclear (PNF) and post-mitochondrial (PMF) fractions, and is not present in significant amounts in mitochondrial or nuclear fractions ( Fig. 1c and 1d).It is possible that a small fraction of mitochondrial or nuclear-targeted sensor may be present in the cytosol and confound analysis of sub-compartmental heme. In order to assess if this were the case, we permeabilized heme-deficient cells lacking HEM1, which encodes the first enzyme in the heme biosynthetic pathway, ALAS, with digitonin, a mild non-ionic detergent that selectively permeabilizes the plasma membrane but not mitochondrial or nuclear membranes, and treated cells with heme. As indicated in Fig. 1e, only cells expressing cytosolic HS1 exhibited a significant heme-dependent reduction in eGFP/mKATE2 fluorescence ratio, with no significant perturbations to the fluorescence of mitochondrial and nuclear-targeted HS1. These data indicate that mitochondrial and nuclear-targeted HS1 is unresponsive to cytosolic heme. Thus, altogether, our data indicate that cytosolic, nuclear, and mitochondrial heme can be robustly monitored in vivo using HS1.Inter-compartmental heme trafficking rates are monitored by: a. inhibiting heme synthesis with 500 μ M succinylacetone (SA), an inhibitor of PBGS, for ~16 hours in sensor expressing cells; b. removing the block in heme synthesis by re-suspending cells into media lacking SA; and c. monitoring the time-dependent change in the eGFP/mKATE2 ratio (R) of HS1 localized to different locations upon the re-initiation of heme synthesis ( Fig. 2a-d). As described in the Materials and Methods section and Equatio...