Targeting of Tsc13p to nucleus-vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic endoplasmic reticulum (ER) membrane protein that accumulates at nucleus-vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-Delta mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast.     

Nucleus-vacuole junctions and piecemeal microautophagy of the nucleus in S. cerevisiae

Various modes of autophagy conspire to degrade virtually every compartment of the eukaryotic cell. In Saccharomyces cerevisiae, a process called "piecemeal microautophagy of the nucleus" (PMN) even pinches off and degrades nonessential portions of the nucleus. PMN is a constitutive process induced to high levels by starvation or rapamycin, an inhibitor of TOR kinase. PMN occurs at nucleus-vacuole (NV) junctions, which are Velcro-like patches formed by interactions between the vacuole membrane protein Vac8p and the outer-nuclear-membrane protein Nvj1p. In response to nutrient depletion, Nvj1p increasingly binds and sequesters two proteins with roles in lipid metabolism, Osh1p and Tsc13p. Tsc13p is required for the normal biogenesis of PMN vesicles. The sequestration of Osh1p by Nvj1p likely serves to negatively regulate the trafficking of tryptophan permease(s) to the plasma membrane. Thus, NV junctions and PMN orchestrate novel and sophisticated responses to nutrient limitation.     

Microautophagy of the nucleus coincides with a vacuolar diffusion barrier at nuclear-vacuolar junctions

Nuclei bind yeast vacuoles via nucleus-vacuole (NV) junctions. Under nutrient restriction, NV junctions invaginate and release vesicles filled with nuclear material into vacuoles, resulting in piecemeal microautophagy of the nucleus (PMN). We show that the electrochemical gradient across the vacuolar membrane promotes invagination of NV junctions. Existing invaginations persist independently of the gradient, but final release of PMN vesicles requires again V-ATPase activity. We find that NV junctions form a diffusion barrier on the vacuolar membrane that excludes V-ATPase but is enriched in the VTC complex and accessible to other membrane-integral proteins. V-ATPase exclusion depends on the NV junction proteins Nvj1p,Vac8p, and the electrochemical gradient. It also depends on factors of lipid metabolism, such as the oxysterol binding protein Osh1p and the enoyl-CoA reductase Tsc13p, which are enriched in NV junctions, and on Lag1p and Fen1p. Our observations suggest that NV junctions form in two separable steps: Nvj1p and Vac8p suffice to establish contact between the two membranes. The electrochemical potential and lipid-modifying enzymes are needed to establish the vacuolar diffusion barrier, invaginate NV junctions, and form PMN vesicles.     

Piecemeal microautophagy of the nucleus: Genetic and morphological traits

Nucleus-vacuole (NV) junctions are formed in Saccharomyces cerevisiae through interactions between Vac8 in the vacuole membrane and Nvj1 in the perinuclear ER. Upon starvation, vesicles containing part of the nucleus emanate from these contact sites and finally pinch off into invaginations of the vacuole. Due to its morphological similarity to microautophagy this process had been termed "piecemeal microautophagy of the nucleus" (PMN). We recently discovered that a number of ATG genes required for macroautophagy and micropexophagy are also required for PMN and accordingly named it micronucleophagy. Therefore, PMN represents a novel model system to investigate the functions of the highly conserved but poorly understood core autophagic apparatus. We here extend the morphological analysis of PMN using immunogold and freeze fracture electron microscopy.     

Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including beta-catenin and importin alpha, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus-vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40-60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in "orphan" rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Delta cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Delta and vac8-Delta cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.     

Piecemeal microautophagy of the nucleus requires the core macroautophagy genes

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.     

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.     

A late form of nucleophagy in Saccharomyces cerevisiae

Autophagy encompasses several processes by which cytosol and organelles can be delivered to the vacuole/lysosome for breakdown and recycling. We sought to investigate autophagy of the nucleus (nucleophagy) in the yeast Saccharomyces cerevisiae by employing genetically encoded fluorescent reporters. The use of such a nuclear reporter, n-Rosella, proved the basis of robust assays based on either following its accumulation (by confocal microscopy), or degradation (by immunoblotting), within the vacuole. We observed the delivery of n-Rosella to the vacuole only after prolonged periods of nitrogen starvation. Dual labeling of cells with Nvj1p-EYFP, a nuclear membrane reporter of piecemeal micronucleophagy of the nucleus (PMN), and the nucleoplasm-targeted NAB35-DsRed.T3 allowed us to detect PMN soon after the commencement of nitrogen starvation whilst delivery to the vacuole of the nucleoplasm reporter was observed only after prolonged periods of nitrogen starvation. This later delivery of nuclear components to the vacuole has been designated LN (late nucleophagy). Only a very few cells showed simultaneous accumulation of both reporters (Nvj1p-EYFP and NAB35-DsRed.T3) in the vacuole. We determined, therefore, that delivery of the two respective nuclear reporters to the vacuole is temporally and spatially separated. Furthermore, our data suggest that LN is mechanistically distinct from PMN because it can occur in nvj1Δ and vac8Δ cells, and does not require ATG11. Nevertheless, a subset of the components of the core macroautophagic machinery is required for LN as it is efficiently inhibited in null mutants of several autophagy-related genes (ATG) specifying such components. Moreover, the inhibition of LN in some mutants is accompanied by alterations in nuclear morphology.     

Measuring piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae

Piecemeal microautophagy of the nucleus (PMN) selectively removes and degrades small fragments of the Saccharomyces cerevisiae nucleus. Inter-organelle contact sites called nucleus-vacuole (NV) junctions determine the selectivity of PMN by establishing a platform for the biogenesis of PMN blebs and vesicles. PMN structures can be observed by fluorescence microscopy using GFP-tagged reporters; however, this approach is best supported with quantitative immunoblot assays of PMN-specific cargo degradation. Together, these assays should facilitate the further study of this fascinating but poorly understood autophagic process in different genetic backgrounds, physiological states, and environmental conditions.     

Why just eat in,when you can also eat out?

The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).     

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.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Nucleocytoplasmic shuttling of the Cdc42p exchange factor Cdc24p

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle-dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p-Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p-Far1p complex which is required for chemotropism.     

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast.

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

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.     

The Vacuole Import and Degradation Pathway Utilizes Early Steps of Endocytosis and Actin Polymerization to Deliver Cargo Proteins to the Vacuole for Degradation

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.     

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.     

Starvation-induced degradation of yeast hexose transporter Hxt7p is dependent on endocytosis, autophagy and the terminal sequences of the permease

The yeast high-affinity glucose transporters Hxt6p and Hxt7p are rapidly degraded during nitrogen starvation in the presence of high concentrations of fermentable carbon sources. Our results suggest that degradation is mainly due to the stimulation of general protein turnover and not caused by a mechanism specifically triggered by glucose. Analysis of Hxt6p/7p stability and cellular distribution in end4, aut2 and apg1 mutants indicates that Hxt7p is internalized by endocytosis, and autophagy is involved in the final delivery of Hxt7p to the vacuole for proteolytic degradation. Internalization and degradation of Hxt7p were blocked after truncation of its N-terminal hydrophilic domain. Nevertheless, this fully functional and stabilized hexose transporter could not maintain fermentation capacity of the yeast cells under starvation conditions, indicating a regulatory constraint on glucose uptake.