Charakterisierung der späten Schritte des peroxisomalen Matrixprotein Imports in Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

Im Rahmen der vorliegenden Arbeit ist eine funktionelle Analyse der späten Schritte des peroxisomalen Matrixproteinimports in $\textit {Saccharomyces cerevisiae}$ durchgeführt worden. Die Hauptaussagen können wie folgt zusammengefasst werden: 1) Bei Deletion oder Inaktivierung der Komponenten des AAA- oder des Pex4p/Pex22p-Komplexes wird der PTS1-Rezeptor Pex5p von Ubc4p an zwei Lysinresten polyubiquitinyliert und anschließend im Proteasom degradiert. 2) Mittels der Etablierung eines $\textit {in vitro}$ Export Systems konnte den AAA-Peroxinen Pex1p und Pex6p die Funktion als Dislokasen für den ATP-abhängigen Export von Pex5p zugewiesen werden. 3) Für Pex4p konnte Pex5p als Substrat für eine Monoubiquitinylierung identifiziert werden. 4) Der Energieabhängigkeit des peroxisomalen Proteinimports kann letztendlich in zwei Schritte differenziert werden: Pex4p-abhängige Rezeptor-Ubiquitinylierung und AAA-abhängige Dislokation

The novel peroxin Pex37

Peroxisomes can undergo fission during cell division, followed by their segregation between mother and daughter cells. Despite species‐specific variations in the molecular composition of the fission machinery, the central mechanistic factors can be assigned to two groups: the Pex11 family and the dynamin‐related protein family. In a recent study, Singh et al. describe the involvement of a member of the Pxmp2‐related protein family in peroxisome fission: the novel peroxin Pex37

Autophagy stimulus-dependent role of the small GTPase Ras2 in peroxisome degradation

The changing accessibility of nutrient resources induces the reprogramming of cellular metabolism in order to adapt the cell to the altered growth conditions. The nutrient-depending signaling depends on the kinases mTOR (mechanistic target of rapamycin), which is mainly activated by nitrogen-resources, and PKA (protein kinase A), which is mainly activated by glucose, as well as both of their associated factors. These systems promote protein synthesis and cell proliferation, while they inhibit degradation of cellular content by unselective bulk autophagy. Much less is known about their role in selective autophagy pathways, which have a more regulated cellular function. Especially, we were interested to analyse the central Ras2-module of the PKA-pathway in the context of peroxisome degradation. Yeast Ras2 is homologous to the mammalian Ras proteins, whose mutant forms are responsible for 33% of human cancers. In the present study, we were able to demonstrate a context-dependent role of Ras2 activity depending on the type of mTOR-inhibition and glucose-sensing situation. When mTOR was inhibited directly via the macrolide rapamycin, peroxisome degradation was still partially suppressed by Ras2, while inactivation of Ras2 resulted in an enhanced degradation of peroxisomes, suggesting a role of Ras2 in the inhibition of peroxisome degradation in glucose-grown cells. In contrast, the inhibition of mTOR by shifting cells from oleate-medium, which lacks glucose, to pexophagy-medium, which contains glucose and is limited in nitrogen, required Ras2-activity for efficient pexophagy, strongly suggesting that the role of Ras2 in glucose sensing-associated signaling is more important in this context than its co-function in mTOR-related autophagy-inhibition

Regulation of the tumor-suppressor function of the class III phosphatidylinositol 3-kinase complex by Ubiquitin and SUMO

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept

mTOR

The mechanistic target of Rapamycin (mTOR) is a ubiquitously-conserved serine/threonine kinase, which has a central function in integrating growth signals and orchestrating their physiologic effects on cellular level. mTOR is the core component of differently composed signaling complexes that differ in protein composition and molecular targets. Newly identified classes of mTOR inhibitors are being developed to block autoimmune diseases and transplant rejections but also to treat obesity, diabetes, and different types of cancer. Therefore, the selective and context-dependent inhibition of mTOR activity itself might come into the focus as molecular target to prevent severe diseases and possibly to extend life span. This review provides a general introduction to the molecular composition and physiologic function of mTOR complexes as part of the Special Issue “2018 Select Papers by $\textit {Cells’}$ Editorial Board Members”

Autophagy-related deubiquitinating enzymes involved in health and disease

Autophagy is an evolutionarily-conserved process that delivers diverse cytoplasmic components to the lysosomal compartment for either recycling or degradation. This involves the removal of protein aggregates, the turnover of organelles, as well as the elimination of intracellular pathogens. In this situation, when only specific cargoes should be targeted to the lysosome, the potential targets can be selectively marked by the attachment of ubiquitin in order to be recognized by autophagy-receptors. Ubiquitination plays a central role in this process, because it regulates early signaling events during the induction of autophagy and is also used as a degradation-tag on the potential autophagic cargo protein. Here, we review how the ubiquitin-dependent steps of autophagy are balanced or counteracted by deubiquitination events. Moreover, we highlight the functional role of the corresponding deubiquitinating enzymes and discuss how they might be involved in the occurrence of cancer, neurodegenerative diseases or infection with pathogenic bacteria

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5p$_{C6A}$, Pex5p$_{C6K}$ turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5p$_{C6K}$ displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5p$_{C6K}$ displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p

Regulation of the tumor-suppressor BECLIN 1 by distinct ubiquitination cascades

Autophagy contributes to cellular homeostasis through the degradation of various intracellular targets such as proteins, organelles and microbes. This relates autophagy to various diseases such as infections, neurodegenerative diseases and cancer. A central component of the autophagy machinery is the class III phosphatidylinositol 3-kinase (PI3K-III) complex, which generates the signaling lipid phosphatidylinositol 3-phosphate (PtdIns3P). The catalytic subunit of this complex is the lipid-kinase VPS34, which associates with the membrane-targeting factor VPS15 as well as the multivalent adaptor protein BECLIN 1. A growing list of regulatory proteins binds to BECLIN 1 and modulates the activity of the PI3K-III complex. Here we discuss the regulation of BECLIN 1 by several different types of ubiquitination, resulting in distinct polyubiquitin chain linkages catalyzed by a set of E3 ligases. This contribution is part of the Special Issue "Ubiquitin System"

Fluidity and lipid composition of membranes of peroxisomes, mitochondria and the ER from oleic acid-induced Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

The maintenance of a fluid lipid bilayer is key for organelle function and cell viability. Given the critical role of lipid compositions in determining membrane properties and organelle identity, it is clear that cells must have elaborate mechanism for membrane maintenance during adaptive responses to environmental conditions. Emphasis of the presented study is on peroxisomes, oleic acid-inducible organelles that are essential for the growth of yeast under conditions of oleic acid as single carbon source. Here, we isolated peroxisomes, mitochondria and ER from oleic acid-induced $\textit {Saccharomyces cerevisiae}$ and determined the lipid composition of their membranes using shotgun lipidomics and compared it to lipid ordering using fluorescence microscopy. In comparison to mitochondrial and ER membranes, the peroxisomal membranes were slightly more disordered and characterized by a distinct enrichment of phosphaditylinositol, indicating an important role of this phospholipid in peroxisomal membrane associated processes

Vac8 controls vacuolar membrane dynamics during different autophagy pathways in Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in $\textit {Saccharomyces cerevisiae}$. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the $\it vac\Delta$ strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner