Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.     

Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling

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Ultrastructure of autophagy in plant cells

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.     

ESCRTing endoplasmic reticulum to microautophagic degradation

ABSTRACT

Changing conditions necessitate cellular adaptation, which frequently entails adjustment of organelle size and shape. The endoplasmic reticulum (ER) is an organelle of exceptional morphological plasticity. In budding yeast, ER stress triggers the de novo formation of ER subdomains called ER whorls. These whorls are selectively degraded by a poorly defined type of microautophagy. We recently showed that ESCRT proteins are essential for microautophagic uptake of ER whorls into lysosomes, likely by mediating the final scission of the lysosomal membrane. Furthermore, ER-selective microautophagy acts in parallel with ER-selective macroautophagy. The molecular machineries for these two types of autophagy are distinct and their contributions to ER turnover vary according to conditions, suggesting that they serve different functions. Our study provides evidence for a direct role of ESCRTs in microautophagy and extends our understanding of how autophagy promotes organelle homeostasis.     

Nijmegen breakage syndrome: The clearance pathway for mutant nibrin protein is allele specific

The autosomal recessive disorder Nijmegen breakage syndrome (NBS) is caused by mutations in the NBN gene which codes for the protein nibrin (NBS1; p95). In the majority of cases, a 5 bp deletion, a founder mutation, leads to a hypomorphic 70 kD protein, p70-nibrin, after alternative initiation of translation. Protein levels are of relevance for the clinical course of the disease, particularly with regard to malignancy. Here, mechanisms and efficiency of mutant protein clearance were examined in order to establish whether these have an impact on nibrin abundance. Cell lines from NBS patients and retroviral transductants were treated with proteasome and lysosome inhibitors and examined by semi-quantitative immunoblotting for p70-nibrin and p95-nibrin levels. The results show that p70-nibrin is degraded by the proteasome with varying efficiency in cell lines from different NBS patients leading to lower or higher steady state levels of this partially active protein fragment. In contrast, a previously described NBN missense mutation, which disturbs protein folding due to the substitution of a critical arginine by tryptophan, was found to be cleared by lysosomal microautophagy leading also to lower cellular levels. The data show that truncated nibrin and misfolded nibrin have different clearance pathways.     

The PI3 Kinase Complex II–PI3P–Vps27 Axis on Vacuolar Membranes is Critical for Microautophagy Induction and Nutrient Stress Adaptation

Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII–PI3P–Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.     

The luminal N-terminus of yeast Nvj1 is an inner nuclear membrane anchor

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.     

Endosomal microautophagy is an integrated part of the autophagic response to amino acid starvation

Starvation is a fundamental type of stress naturally occurring in biological systems. All organisms have therefore evolved different safeguard mechanisms to cope with deficiencies in various types of nutrients. Cells, from yeast to humans, typically respond to amino acid starvation by initiating degradation of cellular components by inducing autophagy. This degradation releases metabolic building blocks to sustain essential core cellular processes. Increasing evidence indicates that starvation-induced autophagy also acts to prepare cells for prolonged starvation by degrading key regulators of different cellular processes. In a recent study, we found that within the first hours of amino acid starvation cells elicit an autophagic response causing rapid degradation of specific proteins. The response is executed independently of both MTOR and canonical macroautophagy. Based on RNAi-mediated knockdown of essential components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery and electron microscopy we conclude that the response relies on some sort of endosomal microautophagy, hence vesicle budding into endosomes. Substantiated by the different substrates that are selectively degraded by this novel pathway we propose that the response predominantly acts to prepare cells for prolonged starvation. Intriguingly, this includes shutting down selective macroautophagy in preparation for a massive induction of bulk macroautophagy.     

Post-ER degradation of misfolded GPI-anchored proteins is linked with microautophagy

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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

ABSTRACT

Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19–33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways.