Istvan R. Boldogh

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.

Purification and Subfractionation of Mitochondria from the Yeast Saccharomyces cerevisiae

Publisher Summary This chapter focuses on the well-established protocols for isolating yeast mitochondria, purifying the organelle using Nycodenz gradients, determining the integrity of isolated mitochondria, and fractionating mitochondria into their sub-compartments. It discusses several protocols for isolation of mitochondria such as growth of yeast cells, isolation of crude mitochondria, and purification of crude mitochondria using continuous Nycodenz gradient centrifugation. It describes how these methods may be used to determine whether a protein localizes to mitochondria and to sub-compartments (outer membrane, inner membrane, inter-membrane space, and matrix) within the organelle. Isolated mitochondria can be fractionated further into inner and outer membranes, as well as contact sites, which are sites of close contact between the outer and inner membranes that are implicated as sites for translocation of protein and phospholipids across, and between, the mitochondrial outer and inner membranes. Isolated mitochondria are routinely used to study activities of resident mitochondrial proteins and determine whether a protein localizes to mitochondria. For both applications, it is critical to document the purity and integrity of the mitochondrial preparation. The simplest way to do so is to perform SDS-PAGE and Western blot analysis, using antibodies raised against proteins of different membrane organelles. The chapter also describes the methods to determine the disposition of proteins on mitochondrial membranes.

Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

Fluorescence loss in photobleaching experiments and analysis of mitochondrial function using superoxide and redox potential biosensors revealed that mitochondria within individual yeast cells are physically and functionally distinct. Mitochondria that are retained in mother cells during yeast cell division have a significantly more oxidizing redox potential and higher superoxide levels compared to mitochondria in buds. Retention of mitochondria with more oxidizing redox potential in mother cells occurs to the same extent in young and older cells and can account for the age‐associated decline in total cellular mitochondrial redox potential in yeast as they age from 0 to 5 generations. Deletion of Mmr1p, a member of the DSL1 family of tethering proteins that localizes to mitochondria at the bud tip and is required for normal mitochondrial inheritance, produces defects in mitochondrial quality control and heterogeneity in replicative lifespan (RLS). Long‐lived mmr1Δ cells exhibit prolonged RLS, reduced mean generation times, more reducing mitochondrial redox potential and lower mitochondrial superoxide levels compared to wild‐type cells. Short‐lived mmr1Δ cells exhibit the opposite phenotypes. Moreover, short‐lived cells give rise exclusively to short‐lived cells, while the majority of daughters of long‐lived cells are long lived. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower‐functioning mitochondria in mother cells and that this process contributes to both mother–daughter age asymmetry and age‐associated declines in cellular fitness.

Role for cER and Mmr1p in Anchorage of Mitochondria at Sites of Polarized Surface Growth in Budding Yeast

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function.

Mitochondrial Inheritance in Budding Yeast

During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae. A combination of genetics, cell‐free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division.

Inheritance of the fittest mitochondria in yeast

Eukaryotic cells compartmentalize their biochemical processes within organelles, which have specific functions that must be maintained for overall cellular health. As the site of aerobic energy mobilization and essential biosynthetic activities, mitochondria are critical for cell survival and proliferation. Here, we describe mechanisms to control the quality and quantity of mitochondria within cells with an emphasis on findings from the budding yeast Saccharomyces cerevisiae. We also describe how mitochondrial quality and quantity control systems that operate during cell division affect lifespan and cell cycle progression.

Taking the A-train: actin-based force generators and organelle targeting

The actin-driven process of cytoplasmic streaming in plant cells is widely believed to be the earliest documented example of cytoskeleton-driven organelle movement. In the decades since these seminal findings, two mechanisms of actin-based intracellular movement have been identified in multiple cell types: one is myosin dependent and the other is dependent upon the Arp2/3 complex for actin nucleation and polymerization. Here, we describe mechanisms of force generation and directed movement that use the actin cytoskeleton, as well as those that target actin-dependent force generators to different subcellular compartments.

Mitochondrial inheritance is required for MEN-regulated cytokinesis in budding yeast

Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p, and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Delta gem1Delta) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of beta-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile-ring closure, but does not inhibit contractile-ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation, or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Delta cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Delta and mmr1Delta gem1Delta cells and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Delta cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

Loss of mitochondrial DNA activates the DNA damage checkpoint kinase Rad53 to inhibit G1- to S-phase progression in budding yeast.

Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris

Production of recombinant proteins is affected by process conditions, where transcriptional regulation of Pichia pastoris alcohol oxidase 1 (PpAOX1) promoter has been a key factor to influence expression levels of proteins of interest. Here, we demonstrate that the AOX1 promoter and peroxisome biogenesis are regulated based on different process conditions. Two types of GFP-fusion proteins, Ub-R-GFP (short-lived GFP in the cytosol) and GFP-SKL (peroxisomal targeting GFP), were successfully used to characterize the time-course of the AOX1 promoter and peroxisome biogenesis, respectively. The activity of the AOX1 promoter and peroxisome biogenesis was highly subjected to different fermentation process conditions - methanol-limited condition at normoxy (ML), switched feeding of carbon sources (e.g., glucose and methanol) under carbon-limited condition at normoxy (SML), and oxygen-limited (OL) condition. The AOX1 promoter was most active under the ML, but less active under the OL. Peroxisome biogenesis showed a high dependency on methanol consumption. In addition, the proliferation of peroxisomes was inhibited in a medium containing glucose and stimulated in the methanol phase under a carbon-limited fed-batch culture condition. The specific productivity of a monoclonal antibody (qp) under the AOX1 promoter was higher at 86h of induction in the ML than in the OL (0.026 vs 0.020mgg(-1)h(-1)). However, the oxygen-limited condition was a robust process suitable for longer induction (180h) due to high cell fitness. Our study suggests that the maximal production of a recombinant protein is highly dependent on methanol consumption rate that is affected by the availability of methanol and oxygen molecules.