A designed point mutant in Fis1 disrupts dimerization and mitochondrial fission.

Mitochondrial and peroxisomal fission are essential processes with defects resulting in cardiomyopathy and neonatal lethality. Central to organelle fission is Fis1, a monomeric tetratricopeptide repeat (TPR)-like protein whose role in assembly of the fission machinery remains obscure. Two nonfunctional, Saccharomyces cerevisiae Fis1 mutants (L80P or E78D/I85T/Y88H) were previously identified in genetic screens. Here, we find that these two variants in the cytosolic domain of Fis1 (Fis1ΔTM) are unexpectedly dimeric. A truncation variant of Fis1ΔTM that lacks an N-terminal regulatory domain is also found to be dimeric. The ability to dimerize is a property innate to the native Fis1ΔTM amino acid sequence as we find this domain is dimeric after transient exposure to elevated temperature or chemical denaturants and is kinetically trapped at room temperature. This is the first demonstration of a specific self-association in solution for the Fis1 cytoplasmic domain. We propose a three-dimensional domain-swapped model for dimerization that is validated by a designed mutation, A72P, which potently disrupts dimerization of wild-type Fis1. A72P also disrupts dimerization of nonfunctional variants, indicating a common structural basis for dimerization. The obligate monomer variant A72P, like the dimer-promoting variants, is nonfunctional in fission, consistent with a model in which Fis1 activity depends on its ability to interconvert between monomer and dimer species. These studies suggest a new functionally important manner in which TPR-containing proteins may reversibly self-associate.

Mon2 is a negative regulator of the monomeric G protein, Arl1.

Using site-directed mutants of ARL1 predicted to alter nucleotide binding, we examined phenotypes associated with the loss of ARL1 , including effects on membrane traffic and K (+) homeostasis. The GTP-restricted allele, ARL[Q72L] , complemented the membrane traffic phenotype (CPY secretion), but not the K (+) homeostasis phenotypes (sensitivity to hygromycin B, steady-state levels of K (+) , and accumulation of (86) Rb (+) ), while the XTP-restricted mutant, ARL1[D130N] , complemented the ion phenotypes, but not the membrane traffic phenotype. A GDP-restricted allele, ARL1[T32N] , did not effectively complement either phenotype. These results are consistent with a model in which Arl1 has three different conformations in vivo. We also explored the relationship between ARL1 and MON2 using the synthetic lethal phenotype exhibited by these two genes and demonstrated that MON2 is a negative regulator of the GTP-restricted allele of ARL1 , ARL1[Q72L] . Finally, we constructed several new alleles predicted to alter binding of Arl1 to the sole GRIP domain containing protein in yeast, Imh1, and found that ARL1[F52G] and ARL1[Y82G] were unable to complement the loss of ARL1 with respect to either the membrane traffic or K (+) homeostasis phenotypes. Our study expands understanding of the roles of Arl1 in vivo.

A lethal de novo mutation in the middle domain of the dynamin-related GTPase Drp1 impairs higher order assembly and mitochondrial division.

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736-1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.

Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis.

The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulates HAT activity. Here we have developed a strategy to introduce acetyl-Lys at up to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA substrate analogue and that HAT activity correlates positively with degree of acetylation. This study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.

CED-9 and mitochondrial homeostasis in C. elegans muscle.

Mitochondrial homeostasis reflects a dynamic balance between membrane fission and fusion events thought essential for mitochondrial function. We report here that altered expression of the C. elegans BCL2 homolog CED-9 affects both mitochondrial fission and fusion. Although striated muscle cells lacking CED-9 have no alteration in mitochondrial size or ultrastructure, these cells appear more sensitive to mitochondrial fragmentation. By contrast, increased CED-9 expression in these cells produces highly interconnected mitochondria. This mitochondrial phenotype is partially suppressed by increased expression of the dynamin-related GTPase DRP-1, with suppression dependent on the BH3 binding pocket of CED-9. This suppression suggests that CED-9 directly regulates DRP-1, a model supported by our finding that CED-9 activates the GTPase activity of human DRP1. Thus, CED-9 is capable of regulating the mitochondrial fission-fusion cycle but is not essential for either fission or fusion.

Ability of Sit4p to promote K+ efflux via Nha1p is modulated by Sap155p and Sap185p.

We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomyces cerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of SAP185 increases efflux, whereas deletion decreases efflux. Two other homologs, SAP4 and SAP190, are without effect on K+ homeostasis. Both SAP155 and SAP185 require the presence of SIT4 for function, which encodes a PP2A-like phosphatase important for the G1-S transition through the cell cycle. Overexpression of either the outwardly rectifying K+ channel, Tok1p, or the putative plasma membrane K+/H+ antiporter, Kha1p, increases efflux in both wild-type and sit4Delta strains. However, overexpression of the Na+-K+/H+ antiporter, Nha1p, is without effect in a sit4Delta strain, suggesting that Sit4p signals to Nha1p. In summary, the combined activities of Sap155p and Sap185p appear to control the function of Nha1p in K+ homeostasis via Sit4p.

The yeast genes, ARL1 and CCZ1, interact to control membrane traffic and ion homeostasis.

The yeast ARL1 gene, encoding a guanine-nucleotide binding protein of the Arf-like family, exhibits a synthetic genetic interaction with CCZ1. An arl1 Delta ccz1 Delta double mutant was viable but grew slowly, was more sensitive to caffeine, Ca(2+), Zn(2+), and hygromycin B than either single mutant, and had a more severe vacuolar protein sorting phenotype. Overexpression of ARL1 did not suppress ccz1 Delta mutant phenotypes, nor did overexpression of CCZ1 suppress arl1 Delta mutant phenotypes. We conclude that ARL1 and CCZ1 independently contribute to both ion homeostasis and protein sorting.