The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria

Abstract

Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5′-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial ‘PLP-ome’) was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1+) and knockout (MTM1−) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration.

Evidence that COG0325 proteins are involved in PLP homeostasis

“…Because the free PLP pool is only a small proportion of the total pool (di Salvo et al, 2011), changes in the free pool will not be detected with this method; hence, it is still to be determined if the absence of yggS affects the free PLP pool. Methods to measure the PLP-ome or the PLP bound to proteins exist but they are all semi-quantitative (Whittaker et al, 2015). We still decided to compare the bound PLP (PLP to proteins) in the WT and DyggS strains using an antibody that detects PLP bound to proteins (Whittaker et al, 2015).…”

“…Methods to measure the PLP-ome or the PLP bound to proteins exist but they are all semi-quantitative (Whittaker et al, 2015). We still decided to compare the bound PLP (PLP to proteins) in the WT and DyggS strains using an antibody that detects PLP bound to proteins (Whittaker et al, 2015). The soluble fractions of both strains were separated by 2D gels (Fig.…”

In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1

“…Moreover, eukaryotic membrane proteins are fragile molecules once extracted from the membrane and the harsh detergents used for solubilization and purification of the carriers using this method cast doubt about the integrity of the proteins throughout such preparations. Accordingly, biophysical data on interactions between the carriers and their respective substrates exist for only a few MCs (24)(25)(26)(27).…”

Intracellular trafficking of the pyridoxal cofactor. Implications for health and metabolic disease

“…Mitochondrial branched chain amino transferase (BCAT, Bat1p)[39,40] can be overexpressed in yeast mitochondria, which become loaded with high levels of the Bat1p apoprotein as protein import overwhelms PLP availability in that compartment (Figure 3, B&C). Mitochondria can be isolated by standard methods [41,42] and used for in organello uptake assays, fixing the localization of PLP at the endpoint by borohydride reduction of protein conjugates [43]. Affinity purification of tagged mBAT-TwinStrep (Figure 3B) then allows a direct read-out of the conjugated pyridoxine content by combined fluorescence and absorption measurements (Figure 3C).…”

“…The aim of a complete PLP-ome proteomics analysis would to determine the distribution of PLP over the protein population, identifying binding sites and evaluating the degree of cofactor loading. Although the PLP cofactor is labile, PLP-protein adducts can be conveniently stabilized by borohydride reduction, converting the PLP imine (Schiff base) linkage to a hydrolytically stable amine [43]. The covalently-conjugated cofactor in the product remains attached through typical procedures for protein separation and analysis, allowing detailed characterization of the complex.…”