Donna M. Gordon

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.

Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p

Kip1p of Saccharomyces cerevisiae is a bipolar kinesin in the conserved bimC kinesin subfamily that mediates mitotic spindle–pole separation. Here, we show that Kip1p is regulated immediately after anaphase initiation by its rapid degradation. Degradation required the ubiquitin protein ligase called the anaphase-promoting complex, the anaphase-promoting complex activating protein Cdc20, and a unique 43-aa sequence in Kip1p. Degradation also required import of Kip1p into the nucleus, but occurred independently of spindle association. A mutation that stabilized Kip1p impaired anaphase progression. The timing of degradation suggests that Kip1p functions primarily during spindle assembly and metaphase, and that Kip1p degradation facilitates structural changes in the mitotic spindle as anaphase progresses.

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly

Background: The cysteine desulfurase Nfs1 provides sulfur for Fe-S cluster biogenesis in mitochondria. Results: Frataxin or a mutant Fe-S scaffold protein (Isu1Sup) with frataxin-bypassing ability stimulates cysteine binding to Nfs1. Conclusion: Frataxin or Isu1Sup likely induces a conformational change in Nfs1, exposing substrate-binding sites. Significance: Data presented here may help develop a drug for treating Friedreich ataxia associated with frataxin deficiency. For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

The Kinesin-related Protein Kip1p of Saccharomyces cerevisiae Is Bipolar

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.

A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Δ1-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively “zippered” together, forming long stretches in which they are in close contact (Schülke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A.94, 7314–7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.

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.

GTP in the mitochondrial matrix plays a crucial role in organellar iron homoeostasis

Mitochondria are the major site of cellular iron utilization for the synthesis of essential cofactors such as iron-sulfur clusters and haem. In the present study, we provide evidence that GTP in the mitochondrial matrix is involved in organellar iron homoeostasis. A mutant of yeast Saccharomyces cerevisiae lacking the mitochondrial GTP/GDP carrier protein (Ggc1p) exhibits decreased levels of matrix GTP and increased levels of matrix GDP [Vozza, Blanco, Palmieri and Palmieri (2004) J. Biol. Chem. 279, 20850-20857]. This mutant (previously called yhm1) also manifests high cellular iron uptake and tremendous iron accumulation within mitochondria [Lesuisse, Lyver, Knight and Dancis (2004) Biochem. J. 378, 599-607]. The reason for these two very different phenotypic defects of the same yeast mutant has so far remained elusive. We show that in vivo targeting of a human nucleoside diphosphate kinase (Nm23-H4), which converts ATP into GTP, to the matrix of ggc1 mutants restores normal iron regulation. Thus the role of Ggc1p in iron metabolism is mediated by effects on GTP/GDP levels in the mitochondrial matrix.

A GTP-dependent “Push” Is Generally Required for Efficient Protein Translocation across the Mitochondrial Inner Membrane into the Matrix

Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This translocation process requires a membrane potential (ΔΨ) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schülke, N., and Pain, D. (1998) J. Biol. Chem. 273, 1420–1424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of ΔΨ but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of ΔΨ but is dependent on a GTP-mediated “push.” This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cisGTP-dependent push and a transATP-dependent “pull.”

Self-association and precursor protein binding of Saccharomyces cerevisiae Tom40p, the core component of the protein translocation channel of the mitochondrial outer membrane

The precursor protein translocase of the mitochondrial outer membrane (Tom) is a multi-subunit complex containing receptors and a general import channel, of which the core component is Tom40p. Nuclear-encoded mitochondrial precursor proteins are first recognized by surface receptors and then pass through the import channel. The Tom complex has been purified; however, the protein-protein interactions that drive its assembly and maintain its stability have been difficult to study. Here we show that Saccharomyces cerevisiae Tom40p expressed in bacteria and purified to homogeneity associates efficiently with itself. The self-association is very strong and can withstand up to 4 M urea or 1 M salt. The tight self-association does not require the N-terminal segment of Tom40p. Furthermore, purified Tom40p preferentially recognizes the targeting sequence of mitochondrial precursor proteins. Although the binding of the targeting sequence to Tom40p is inhibited by urea concentrations in excess of 1 M, it is moderately resistant to 1 M salt. Simultaneous self-assembly and precursor protein binding suggest that Tom40p contains at least two different domains mediating these processes. The experimental approach described here should be useful for analysing protein-protein interactions involving individual or groups of components of the mitochondrial import machinery.

Occidiofungin's Chemical Stability and In vitro Potency Against Candida species

ABSTRACT Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between 0.5 and 2.0 μg/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal concentration (MLC) of 2 and 4 μg/ml, respectively. Time-kill and postantifungal effect (PAFE) experiments of occidiofungin against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also found to be a very stable molecule. It is resistant to extreme temperatures and pH and maintains its activity following exposure to gastric proteases.