Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic ...compartment, and release the cargo for degradation. The mechanism of autophagosome-vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome-vacuole fusion. We found that this process requires the Atg14-Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome-vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process.
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.
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.
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•Human mitochondrial high-confidence proteome with >1,100 proteins (MitoCoP)•Mitochondria-specific protein copy numbers and half-lives•Interactors of protein translocases and oxidative phosphorylation assembly factors•>40% of mitochondrial proteome linked to human diseases
Mitochondria are crucial for cellular energy metabolism and human health. Morgenstern et al. present a high-confidence protein compendium of human mitochondria including mitochondria-specific protein copy numbers and half-lives. They identify interactors of key mitochondrial protein machineries and link >40% of the mitochondrial proteome to human diseases.
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.
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•Dual role of Mic10 of mitochondrial contact site and cristae organizing system (MICOS)•Mic10 binds to mitochondrial ATP synthase and stabilizes higher order assemblies•Oligomerization of Mic10 is required for its function in MICOS, not at ATP synthase•Mic10 binding to ATP synthase supports metabolic adaptation and respiratory growth
Rampelt et al. report that the core component Mic10 of the mitochondrial contact site and cristae organizing system plays a second physiologically important role at the mitochondrial ATP synthase. Mic10 variants that selectively function at the ATP synthase promote efficient metabolic adaptation, respiratory growth, and mitochondrial inner membrane energization.
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.
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•Nucleotide composition and secondary structures determine mRNA targeting•The SecYEG translocon and the YidC insertase act as mRNA receptors in bacteria•mRNA targeting enables SRP-independent membrane protein insertion
Protein targeting is generally believed to depend on targeting information that is retained within the protein itself. Sarmah et al. now show that mRNA targeting contributes to membrane protein insertion in bacteria and that it provides an alternative strategy when the canonical signal recognition particle-dependent protein-targeting pathway is saturated or inhibited.
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.
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.
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.
The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for ...proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
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•Impaired presequence processing leads to precursor aggregation inside mitochondria•Intramitochondrial precursor aggregates trigger early transcriptional stress response•Relocalization of nuclear transcription factor Rox1 to mitochondria ensures survival•Mitochondrial Rox1 maintains mitochondrial genome expression upon early mtUPR
N-terminal presequences direct cytosolic precursor proteins to mitochondria. Poveda-Huertes et al. show that impaired presequence cleavage leads to proteotoxic aggregates inside mitochondria that trigger an early mtUPR-like stress response. Relocalization of the nuclear transcription factor Rox1 to mitochondria allows maintenance of mtDNA expression ensuring proteostasis and survival upon early mtUPR.
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.
Synopsis
A proteomics approach identifies 48 phosphorylation sites on Atg13, crucial for regulating bulk autophagy initiation. Disrupting Atg13’s dynamic phosphorylation leads to harmful autophagic outcomes, emphasizing the importance of precise post-translational control in regulating bulk autophagy activity.
Extensive post-translational regulation on Atg13 is important early during autophagy initiation.
Disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy.
Atg11 is involved in bulk autophagy also during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site.
A proteomics approach identifies 48 phosphorylation sites on Atg13, crucial for regulating bulk autophagy initiation. Disrupting Atg13’s dynamic phosphorylation leads to harmful autophagic outcomes, emphasizing the importance of precise post-translational control in regulating bulk autophagy activity.
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