Chapter 14 Nucleotide-dependent iron-sulfur cluster biogenesis of endogenous and imported apoproteins in isolated intact mitochondria.

Iron-sulfur [Fe-S] clusters are cofactors of proteins involved in electron transfer, enzyme catalysis, radical generation, sulfur donation, and signal transduction. Biogenesis of [Fe-S] clusters is mediated by numerous conserved proteins present in E. coli and in mitochondria of eukaryotic cells such as yeast and humans. Although a completely reconstituted system for study of this process does not yet exist, isolated intact mitochondria are capable of synthesizing new [Fe-S] clusters when supplied with a few ingredients. Here we describe methods for studying the biogenesis of [Fe-S] clusters in intact mitochondria. In these assays, metabolically active mitochondria isolated from a wild-type Saccharomyces cerevisiae strain are incubated with (35)S-cysteine. The (35)S is rapidly (approximately 15 min) and efficiently incorporated by physiologic pathways into newly formed [Fe-S] clusters and inserted into target proteins. Proteins labeled with [Fe-(35)S] clusters are then separated by native polyacrylamide gel electrophoresis followed by autoradiography, thereby allowing direct visualization and quantitation. Both endogenous (Aco1p aconitase) and newly imported (Yah1p ferredoxin) apoproteins can be used as substrates. [Fe-S] cluster biogenesis in isolated intact mitochondria is greatly enhanced by the addition of nucleotides (GTP and ATP) and requires hydrolysis of both. A major advantage of the methods described here is that neither in vivo overexpression of target substrates nor enrichment by immunoprecipitation is necessary to detect radiolabeled proteins. It is also not necessary to perform these assays under anaerobic conditions, because intact mitochondria are capable of protecting newly formed [Fe-S] clusters from oxidative damage.

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis.

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Delta mrs3 and Delta mrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX(2)CXC and twin CX(2)C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Deltadre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.

GTP is required for iron-sulfur cluster biogenesis in mitochondria.

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

A GTP:AMP phosphotransferase, Adk2p, in Saccharomyces cerevisiae. Role of the C terminus in protein folding/stabilization, thermal tolerance, and enzymatic activity.

Adenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3' end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103-31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30 degrees C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5-10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions.

A novel role of Mgm1p, a dynamin-related GTPase, in ATP synthase assembly and cristae formation/maintenance.

In Saccharomyces cerevisiae, two mitochondrial inner-membrane proteins play critical roles in organellar morphology. One is a dynamin-related GTPase, Mgm1p, which participates in mitochondrial fusion. Another is Tim11p, which is required for oligomeric assembly of F1Fo-ATP synthase, which generates ATP through oxidative phosphorylation. Our data bring these findings together and define a novel role for Mgm1p in the formation and maintenance of mitochondrial cristae. We show that Mgm1p serves as an upstream regulator of Tim11p protein stability, ATP synthase assembly, cristae morphology and cytochrome c storage within cristae.

Nucleoside diphosphate kinase of Saccharomyces cerevisiae, Ynk1p: localization to the mitochondrial intermembrane space.

Nucleoside diphosphate kinase (NDPK) is a highly conserved multifunctional enzyme. It catalyses the transfer of gamma phosphates from nucleoside triphosphates to nucleoside diphosphates by a mechanism that involves formation of an autophosphorylated enzyme intermediate. The phosphate is usually supplied by ATP. NDPK activity in different subcellular compartments may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. NDPKs are homo-oligomeric proteins and are predominantly localized in the cytosol. In this paper, we demonstrate that in Saccharomyces cerevisiae a small fraction of total NDPK activity encoded by YNK1 is present in the intermembrane space (IMS) of mitochondria, and the corresponding protein Ynk1p in the IMS represents approx. 0.005% of total mitochondrial proteins. Ynk1p, synthesized as a single gene product, must therefore be partitioned between cytoplasm and mitochondrial IMS fractions. A mechanism for this partitioning is suggested by our observations that interaction with a 40 kDa protein of the translocase of outer mitochondrial membrane (Tom40p), occurs preferentially with unfolded, unphosphorylated forms of Ynk1p. A population of newly translated, but not yet folded or autophosphorylated, Ynk1p intermediates may be imported into the IMS of mitochondria and trapped there by subsequent folding and oligomerization. Within the small volume of the IMS, Ynk1p may be more concentrated and may be required to supply GTP to several important proteins in this compartment.