Cell cycle-linked vacuolar pH dynamics regulate amino acid homeostasis and cell growth.

Amino acid homeostasis is critical for many cellular processes. It is well established that amino acids are compartmentalized using pH gradients generated between organelles and the cytoplasm; however, the dynamics of this partitioning has not been explored. Here we develop a highly sensitive pH reporter and find that the major amino acid storage compartment in Saccharomyces cerevisiae, the lysosome-like vacuole, alkalinizes before cell division and re-acidifies as cells divide.

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation.

Engineering ER-stress dependent non-conventional mRNA splicing.

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity.

Multiple selection filters ensure accurate tail-anchored membrane protein targeting.

Accurate protein localization is crucial to generate and maintain organization in all cells. Achieving accuracy is challenging, as the molecular signals that dictate a protein's cellular destination are often promiscuous. A salient example is the targeting of an essential class of tail-anchored (TA) proteins, whose sole defining feature is a transmembrane domain near their C-terminus.

Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion.

Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested.

The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins.

The accuracy of tail-anchored (TA) protein targeting to the endoplasmic reticulum (ER) depends on the Guided Entry of Tail-Anchored (Get) protein targeting machinery. The fate of TA proteins that become inappropriately inserted into other organelles, such as mitochondria, is unknown. Here, we identify Msp1, a conserved, membrane-anchored AAA-ATPase (ATPase associated with a variety of cellular activities) that localizes to mitochondria and peroxisomes, as a critical factor in a quality control pathway that senses and degrades TA proteins mistargeted to the outer mitochondrial membrane (OMM).

Pharmacological brake-release of mRNA translation enhances cognitive memory.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR).

Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway.

Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro.

Cofilin recruitment and function during actin-mediated endocytosis dictated by actin nucleotide state.

Cofilin is the major mediator of actin filament turnover in vivo. However, the molecular mechanism of cofilin recruitment to actin networks during dynamic actin-mediated processes in living cells and cofilin's precise in vivo functions have not been determined. In this study, we analyzed the dynamics of fluorescently tagged cofilin and the role of cofilin-mediated actin turnover during endocytosis in Saccharomyces cerevisiae.

A FRET-based approach to ratiometric fluorescence detection of hydrogen peroxide.

We report the synthesis, properties, and biological applications of Ratio-Peroxyfluor-1 (RPF1), a new ratiometric fluorescent reporter for hydrogen peroxide. RPF1 is comprised of a two-fluorophore cassette, where the spectral overlap between coumarin donor and fluoran/fluorescein acceptor partners can be controlled by the chemoselective peroxide-mediated deprotection of boronic ester pendants on the acceptor dye. RPF1 features good selectivity for hydrogen peroxide over a variety of reactive oxygen species, including superoxide and nitric oxide, a ca.

Mdv1 interacts with assembled dnm1 to promote mitochondrial division.

The dynamin-related GTPase, Dnm1, self-assembles into punctate structures that are targeted to the outer mitochondrial membrane where they mediate mitochondrial division. Post-targeting, Dnm1-dependent division is controlled by the actions of the WD repeat protein, Mdv1, and the mitochondrial tetratricopeptide repeat-like outer membrane protein, Fis1. Our previous studies suggest a model where at this step Mdv1 functions as an adaptor linking Fis1 with Dnm1.

The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion.

A balance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane-associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p.

The WD repeat protein, Mdv1p, functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission.

Yeast mitochondrial fission is a multistep process during which the dynamin-related GTPase, Dnm1p, assembles into punctate structures that associate with the outer mitochondrial membrane and mediate mitochondrial division. Steps in the Dnm1p-dependent process of fission are regulated by the actions of the WD repeat protein, Mdv1p, and the mitochondrial outer membrane protein, Fis1p. Our previous studies suggested a model where Mdv1p functions to regulate fission at a post-Dnm1p assembly step and Fis1p functions at two distinct steps, at an early point, to regulate Dnm1p assembly, and later, together with Mdv1p, to facilitate Dnm1p-dependent mitochondrial fission.