Michael P. Yaffe

Presenilins Are Enriched in Endoplasmic Reticulum Membranes Associated with Mitochondria

Presenilin-1 (PS1) and -2 (PS2), which when mutated cause familial Alzheimer disease, have been localized to numerous compartments of the cell, including the endoplasmic reticulum, Golgi, nuclear envelope, endosomes, lysosomes, the plasma membrane, and mitochondria. Using three complementary approaches, subcellular fractionation, gamma-secretase activity assays, and immunocytochemistry, we show that presenilins are highly enriched in a subcompartment of the endoplasmic reticulum that is associated with mitochondria and that forms a physical bridge between the two organelles, called endoplasmic reticulum-mitochondria-associated membranes. A localization of PS1 and PS2 in mitochondria-associated membranes may help reconcile the disparate hypotheses regarding the pathogenesis of Alzheimer disease and may explain many seemingly unrelated features of this devastating neurodegenerative disorder.

Prohibitin Family Members Interact Genetically with Mitochondrial Inheritance Components in Saccharomyces cerevisiae

ABSTRACT Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 andPHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.

MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import.

The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.

Mitochondrial DNA inheritance in Saccharomyces cerevisiae.

Respiratory metabolism depends on mitochondrial DNA, yet the mechanisms that ensure the inheritance of the mitochondrial genome are largely obscure. Recent studies with Saccharomyces cerevisiae suggest that distinct factors mediate the active segregation of mitochondrial DNA during mitotic growth. The identification of the proteins required for the maintenance of the mitochondrial genome provides clues to the mechanisms of, and molecular machinery involved in, mitochondrial DNA inheritance.

Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p

Mgm1p is a conserved dynamin-related GTPase required for fusion, morphology, inheritance, and the genome maintenance of mitochondria in Saccharomyces cerevisiae. Mgm1p undergoes unconventional processing to produce two functional isoforms by alternative topogenesis. Alternative topogenesis involves bifurcate sorting in the inner membrane and intramembrane proteolysis by the rhomboid protease Pcp1p. Here, we identify Ups1p, a novel mitochondrial protein required for the unique processing of Mgm1p and for normal mitochondrial shape. Our results demonstrate that Ups1p regulates the sorting of Mgm1p in the inner membrane. Consistent with its function, Ups1p is peripherally associated with the inner membrane in the intermembrane space. Moreover, the human homologue of Ups1p, PRELI, can fully replace Ups1p in yeast cells. Together, our findings provide a conserved mechanism for the alternative topogenesis of Mgm1p and control of mitochondrial morphology.

Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles

Microtubules mediate mitochondrial distribution in the yeast Schizosaccharomyces pombe and many higher eukaryotic cells. In higher eukaryotes, kinesin motor proteins have been shown to transport mitochondria along microtubules, but the nature of the mitochondria–microtubule interactions in S. pombe has not been explored. By time lapse, total internal reflection fluorescence microscopy, or spinning-disk confocal microscopy, mitochondria appeared to be both tethered to ends and bound laterally along the sides of microtubules. Mitochondrial tubules extended and retracted when attached to the tips of elongating or shortening microtubules, respectively, but translocation along established microtubules was never observed. Mitochondria that were not associated with microtubules were largely immobile until they were “captured” by a growing microtubule. In mitotic cells, a portion of the mitochondria was tethered to the spindle-pole bodies and moved to the cellular ends during spindle elongation. This association may be important for organelle inheritance during cell division. Thus, in contrast to kinesin-mediated transport used by higher eukaryotes, mitochondrial motility and distribution in fission yeast are driven largely by microtubule polymerization and the elongation of the mitotic spindle.

A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle.

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

BRO1, A NOVEL GENE THAT INTERACTS WITH COMPONENTS OF THE PKC1P-MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY IN SACCHAROMYCES CEREVISIAE

Yeast cells with mutations in BRO1 display phenotypes similar to those caused by deletion of BCK1, a gene encoding a MEK kinase that functions in a mitogen-activated protein kinase pathway mediating maintenance of cell integrity. bro1 cells exhibit a temperature-sensitive growth defect that is suppressed by the addition of osmotic stabilizers or Ca2+ to the growth medium or by additional copies of the BCK1 gene. At permissive temperatures, bro1 mutants are sensitive to caffeine and respond abnormally to nutrient limitation. A null mutation in BRO1 is synthetically lethal with null mutations in BCK1, MPK1, which encodes a mitogen-activated protein kinase that functions downstream of Bck1p, or PKC1, a gene encoding a protein kinase C homolog that activates Bck1p. Analysis of the isolated BRO1 gene revealed that it encodes a novel, 97-kDa polypeptide which contains a putative SH3 domain-binding motif and is homologous to a protein of unknown function in Caenorhabditis elegans.

On the translocation of proteins across membranes

Many proteins of intracellular organelles are first synthesized in the cytoplasm and are then specifically transferred across the membranes of the organelles. On the assumption that these transfers all occur by the same basic mechanism, we enumerate the rather stringent requirements that the mechanism must satisfy. A unitary molecular mechanism is then proposed that meets these requirements.

The cutting edge of mitochondrial fusion.

Mitochondrial membrane fusion contributes to the shape and distribution of mitochondria in the eukaryotic cytoplasm. Recent work shows that a key component of the fusion reaction, Mgm1p, is activated by a novel mitochondrial protease related to the Drosophila melanogaster signalling protein Rhomboid.