Spatiotemporal regulation of organelle transport by spindle position checkpoint kinase Kin4.

Asymmetric cell division in Saccharomyces cerevisiae involves class V myosin-dependent transport of organelles along the polarised actin cytoskeleton to the emerging bud. Vac17 is the vacuole/lysosome-specific myosin receptor. Its timely breakdown terminates transport and results in the proper positioning of vacuoles in the bud.

Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown.

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates.

Peroxisomal NAD(H) Homeostasis in the Yeast Depends on Two Redox Shuttles and the NAD Carrier, Pmp47.

is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in .

Spindle Position Checkpoint Kinase Kin4 Regulates Organelle Transport in .

Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes.

Characterization of Yeast Peroxisomes: Enrichment of Peroxisomal Fractions and Analysis of β-Oxidation Activity.

Subcellular fractionation approaches have allowed for the identification of various functionally distinct organelles including peroxisomes. The methods enable enrichment of organelles and combined with downstream assays allow for the identification of biochemical functions, composition, and structural characteristics of these compartments. In this chapter, we describe the methods for differential centrifugation and Nycodenz gradients in the yeast Saccharomyces cerevisiae and describe assays for fatty acid β-oxidation in intact cells and in peroxisomal fractions.

Targeted Modifications of the Yeast Genome to Study Peroxisomes.

PCR-based gene targeting enables rapid alteration of the Saccharomyces cerevisiae genome. Here we describe how this method can be applied for directed gene deletions, epitope and fluorescence protein tagging, and conditional gene expression, with a specific focus on peroxisomal proteins.

The dynamin-related protein Vps1 and the peroxisomal membrane protein Pex27 function together during peroxisome fission.

Dynamin-related proteins (Drps) mediate a variety of membrane remodelling processes. The Saccharomyces cerevisiae Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion, and peroxisome fission and breakdown. How Drps, and in particular Vps1, can function at so many different subcellular locations is of interest to our understanding of cellular organisation.

Contribution of domain structure to the function of the yeast DEDD family exoribonuclease and RNase T functional homolog, Rex1.

The 3' exonucleolytic processing of stable RNAs is conserved throughout biology. Yeast strains lacking the exoribonuclease Rex1 are defective in the 3' processing of stable RNAs, including 5S rRNA and tRNA. The equivalent RNA processing steps in are carried out by RNase T.

Blocking Mitophagy Does Not Significantly Improve Fuel Ethanol Production in Bioethanol Yeast Saccharomyces cerevisiae.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene increased the fermentation performance during the brewing of Ginjo sake.

Inhibition of Arabidopsis stomatal development by plastoquinone oxidation.

Stomata are the pores in the epidermal surface of plant leaves that regulate the exchange of water and CO with the environment thus controlling leaf gas exchange. In the model dicot plant Arabidopsis thaliana, the transcription factors SPEECHLESS (SPCH) and MUTE sequentially control formative divisions in the stomatal lineage by forming heterodimers with ICE1. SPCH regulates entry into the stomatal lineage and its stability or activity is regulated by a mitogen-activated protein kinase (MAPK) signaling cascade, mediated by its interaction with ICE1.

The Pex3-Inp1 complex tethers yeast peroxisomes to the plasma membrane.

A subset of peroxisomes is retained at the mother cell cortex by the Pex3-Inp1 complex. We identify Inp1 as the first known plasma membrane-peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria that a contact site tether protein must adhere to. We show that Inp1 is present in the correct subcellular location to interact with both the plasma membrane and peroxisomal membrane and has the structural and functional capacity to be a PM-PER tether.

Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p.

Peroxisomes are eukaryotic organelles that function in numerous metabolic pathways and defects in peroxisome function can cause serious developmental brain disorders such as adrenoleukodystrophy (ALD). Peroxisomal membrane proteins (PMPs) play a crucial role in regulating peroxisome function. Therefore, PMP homeostasis is vital for peroxisome function.

Two NAD-linked redox shuttles maintain the peroxisomal redox balance in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, peroxisomes are the sole site of fatty acid β-oxidation. During this process, NAD is reduced to NADH. When cells are grown on oleate medium, peroxisomal NADH is reoxidised to NAD by malate dehydrogenase (Mdh3p) and reduction equivalents are transferred to the cytosol by the malate/oxaloacetate shuttle.

Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation.

Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins.

Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis.

A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum-derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes.

Multiple pathways for protein transport to peroxisomes.

Peroxisomes are unique among the organelles of the endomembrane system. Unlike other organelles that derive most if not all of their proteins from the ER (endoplasmic reticulum), peroxisomes contain dedicated machineries for import of matrix proteins and insertion of membrane proteins. However, peroxisomes are also able to import a subset of their membrane proteins from the ER.

Evolving models for peroxisome biogenesis.

Significant progress has been made towards our understanding of the mechanism of peroxisome formation, in particular concerning sorting of peroxisomal membrane proteins, matrix protein import and organelle multiplication. Here we evaluate the progress made in recent years. We focus mainly on progress made in yeasts.

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae.

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells.

Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.

Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain.

Atg36: the Saccharomyces cerevisiae receptor for pexophagy.

Eukaryotic cells adapt their organelle composition and abundance according to environmental conditions. Analysis of the peroxisomal membrane protein Pex3 has revealed that this protein plays a crucial role in peroxisome maintenance as it is required for peroxisome formation, segregation and breakdown. Although its function in peroxisome formation and segregation was known to involve its recruitment to the peroxisomal membrane of factors specific for these processes, the role of Pex3 in peroxisome breakdown was unclear until our recent identification of Atg36 as a novel Saccharomyces cerevisiae Pex3-interacting protein.

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum.

NBP (nitrogen-containing bisphosphonate) drugs protect against excessive osteoclast-mediated bone resorption. After binding to bone mineral, they are taken up selectively by the osteoclasts and inhibit the essential enzyme FDPS (farnesyl diphosphate synthase). NBPs inhibit also growth of amoebae of Dictyostelium discoideum in which their target is again FDPS.

Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae.

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane.

Peroxisome biogenesis: recent advances.

In recent years, it has become evident that peroxisomes form part of the endomembrane system. Peroxisomes can form from the ER via a maturation process and they can multiply by growth and division, whereby the ER provides membrane for growth and ongoing fission (Figure 1). Until very recently, it was widely accepted that most peroxisomal membrane proteins (PMPs) insert directly into peroxisomes, whereas a small subset of PMPs traffic via the ER.