Synthetic Dimerization Approaches for In Vivo Studies in Saccharomyces cerevisiae.

Synthetic tethering approaches induced by chemical means offer precise control over protein interactions in cells. They enable the manipulation of when, where, and how proteins interact, making it possible to study their functions, dynamics, and cellular consequences at a molecular level. These methods are versatile, reversible, and adaptable, allowing the dissection of complex cellular processes and the engineering of cellular functions. Here, we describe two chemically induced dimerization systems in the model organism Saccharomyces cerevisiae. Using the autophagy pathway as an example, we show how these approaches can be used to dissect molecular events in cells.

FIP200 Phosphorylation Regulates Late Steps in Mitophagy.

Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

Beyond the C-terminal Glycine of ATG8 Proteins - The Story of Some Neglected Amino Acids.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Phagophore closure, autophagosome maturation and autophagosome fusion during macroautophagy in the yeast Saccharomyces cerevisiae.

Macroautophagy, hereafter referred to as autophagy, is a complex process in which multiple membrane-remodeling events lead to the formation of a cisterna known as the phagophore, which then expands and closes into a double-membrane vesicle termed the autophagosome. During the past decade, enormous progress has been made in understanding the molecular function of the autophagy-related proteins and their role in generating these phagophores. In this Review, we discuss the current understanding of three membrane remodeling steps in autophagy that remain to be largely characterized; namely, the closure of phagophores, the maturation of the resulting autophagosomes into fusion-competent vesicles, and their fusion with vacuoles/lysosomes. Our review will mainly focus on the yeast Saccharomyces cerevisiae, which has been the leading model system for the study of molecular events in autophagy and has led to the discovery of the major mechanistic concepts, which have been found to be mostly conserved in higher eukaryotes.

Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry.

We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria.

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.

Dual role of Mic10 in mitochondrial cristae organization and ATP synthase-linked metabolic adaptation and respiratory growth.

Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric F1Fo-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10ATPsynthase, not on Mic10MICOS. We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F1Fo-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth.

Phosphoregulation of the autophagy machinery by kinases and phosphatases.

Eukaryotic cells use post-translational modifications to diversify and dynamically coordinate the function and properties of protein networks within various cellular processes. For example, the process of autophagy strongly depends on the balanced action of kinases and phosphatases. Highly conserved from the budding yeast Saccharomyces cerevisiae to humans, autophagy is a tightly regulated self-degradation process that is crucial for survival, stress adaptation, maintenance of cellular and organismal homeostasis, and cell differentiation and development. Many studies have emphasized the importance of kinases and phosphatases in the regulation of autophagy and identified many of the core autophagy proteins as their direct targets. In this review, we summarize the current knowledge on kinases and phosphatases acting on the core autophagy machinery and discuss the relevance of phosphoregulation for the overall process of autophagy.

The lipid flippase Drs2 regulates anterograde transport of Atg9 during autophagy.

Macroautophagy/autophagy is a conserved catabolic pathway during which cellular material is sequestered within newly formed double-membrane vesicles called autophagosomes and delivered to the lytic compartment of eukaryotic cells for degradation. Autophagosome biogenesis depends on the core autophagy-related (Atg) machinery, and involves a massive supply and remodelling of membranes. To gain insight into the lipid remodelling mechanisms during autophagy, we have systematically investigated whether lipid flippases are required for this pathway in the yeast Saccharomyces cerevisiae. We found that the flippase Drs2, which transfers phosphatidylserine and phosphatidylethanolamine from the lumenal to the cytosolic leaflet of the limiting membrane at the trans-Golgi network, is required for normal progression of autophagy. We also show that Drs2 is important for the trafficking of the core Atg protein Atg9. Atg9 is a transmembrane protein important for autophagosome biogenesis and its anterograde transport from its post-Golgi reservoirs to the site of autophagosome formation is severely impaired in the absence of Drs2. Thus, our results identify a novel autophagy player and highlight that membrane asymmetry regulates early autophagy steps.

The mechanism of macroautophagy: The movie.

This animated movie presents the mechanism of macroautophagy, hereafter autophagy, by showing the molecular features of the formation of autophagosomes, the hallmark organelle of this intracellular catabolic pathway. It is based on our current knowledge and it also illustrates how autophagosomes can recognize and eliminate selected cargoes.

Spatial control of avidity regulates initiation and progression of selective autophagy.

Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context.

Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery.

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy.

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.

Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling.

Autophagy mediates the degradation of cytoplasmic material. Upon autophagy induction, autophagosomes form a sealed membrane around the cargo and fuse with the lytic compartment to release the cargo for degradation. In order to avoid premature fusion of immature autophagosomal membranes with the lytic compartment, this process needs to be tightly regulated. Several factors mediating autophagosome-vacuole fusion have recently been identified. In budding yeast, autophagosome-vacuole fusion requires the R-SNARE Ykt6 on the autophagosome, together with the three Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. However, how these SNAREs are regulated during the fusion process is poorly understood. In this study, we investigate the regulation of Ykt6. We found that Ykt6 is directly phosphorylated by Atg1 kinase, which keeps this SNARE in an inactive state. Ykt6 phosphorylation prevents SNARE bundling by disrupting its interaction with the vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion. These findings shed new light on the regulation of autophagosome-vacuole fusion and reveal a further step in autophagy controlled by the Atg1 kinase.

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle.

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.