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.

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On The Unique Structural Organization of the Saccharomyces Cerevisiae Pyruvate Dehydrogenase Complex

Microscopy and Microanalysis ◽

10.1017/s1431927600024892 ◽

1998 ◽

Vol 4 (S2) ◽

pp. 954-955

Author(s):

James K. Stoops ◽

Z. Hong Zhou ◽

John P. Schroeter ◽

Steven J. Kolodziej ◽

R. Holland Cheng ◽

...

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Pyruvate Dehydrogenase ◽

Structural Organization ◽

Pyruvate Dehydrogenase Complex ◽

The Other ◽

Oxidative Decarboxylation ◽

Multienzyme Complex ◽

The Core ◽

Dehydrogenase Complex

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

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Key Regulators of Autophagosome Closure

Cells ◽

10.3390/cells10112814 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2814

Author(s):

Wenyan Jiang ◽

Xuechai Chen ◽

Cuicui Ji ◽

Wenting Zhang ◽

Jianing Song ◽

...

Keyword(s):

Recent Progress ◽

Membrane Vesicles ◽

Rab Gtpase ◽

The Core ◽

Escrt Machinery ◽

Atg Proteins ◽

Evolutionarily Conserved ◽

Atg Genes ◽

Related Proteins ◽

Key Questions

Autophagy is an evolutionarily conserved pathway, in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation. Over 40 conserved autophagy-related (ATG) genes define the core machinery for the five processes of autophagy: initiation, nucleation, elongation, closure, and fusion. In this review, we focus on one of the least well-characterized events in autophagy, namely the closure of the isolation membrane/phagophore to form the sealed autophagosome. This process is tightly regulated by ESCRT machinery, ATG proteins, Rab GTPase and Rab-related proteins, SNAREs, sphingomyelin, and calcium. We summarize recent progress in the regulation of autophagosome closure and discuss the key questions remaining to be addressed.

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Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0483 ◽

2003 ◽

Vol 14 (1) ◽

pp. 129-141 ◽

Cited By ~ 270

Author(s):

Paul Roberts ◽

Sharon Moshitch-Moshkovitz ◽

Erik Kvam ◽

Eileen O'Toole ◽

Mark Winey ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Nitrogen Starvation ◽

Nutrient Sensing ◽

Dependent Manner ◽

Vacuole Membrane ◽

Carbon And Nitrogen ◽

Transmission Electron ◽

Biochemical Assays ◽

Dividing Cells ◽

Tor Kinase

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promotepiecemeal microautophagy of thenucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-Δcells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.

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Defining Functions of Mannoproteins in Saccharomyces cerevisiae by High-Dimensional Morphological Phenotyping

Journal of Fungi ◽

10.3390/jof7090769 ◽

2021 ◽

Vol 7 (9) ◽

pp. 769

Author(s):

Farzan Ghanegolmohammadi ◽

Hiroki Okada ◽

Yaxuan Liu ◽

Kaori Itto-Nakama ◽

Shinsuke Ohnuki ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Size ◽

Gaussian Mixture ◽

High Dimensional ◽

Actin Organization ◽

Apical Bud ◽

Biochemical Assays ◽

Morphological Defects ◽

Mutant Cells

Mannoproteins are non-filamentous glycoproteins localized to the outermost layer of the yeast cell wall. The physiological roles of these structural components have not been completely elucidated due to the limited availability of appropriate tools. As the perturbation of mannoproteins may affect cell morphology, we investigated mannoprotein mutants in Saccharomyces cerevisiae via high-dimensional morphological phenotyping. The mannoprotein mutants were morphologically classified into seven groups using clustering analysis with Gaussian mixture modeling. The pleiotropic phenotypes of cluster I mutant cells (ccw12Δ) indicated that CCW12 plays major roles in cell wall organization. Cluster II (ccw14Δ, flo11Δ, srl1Δ, and tir3Δ) mutants exhibited altered mother cell size and shape. Mutants of cluster III and IV exhibited no or very small morphological defects. Cluster V (dse2Δ, egt2Δ, and sun4Δ) consisted of endoglucanase mutants with cell separation defects due to incomplete septum digestion. The cluster VI mutant cells (ecm33Δ) exhibited perturbation of apical bud growth. Cluster VII mutant cells (sag1Δ) exhibited differences in cell size and actin organization. Biochemical assays further confirmed the observed morphological defects. Further investigations based on various omics data indicated that morphological phenotyping is a complementary tool that can help with gaining a deeper understanding of the functions of mannoproteins.

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The Structure and Development of Microbodies in the Fat Body and other Tissues of an Insect (Calpodes Ethlius)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006773x ◽

1970 ◽

Vol 28 ◽

pp. 148-149

Author(s):

M. Locke ◽

J. T. McMahon

Keyword(s):

Electron Microscopy ◽

Liquid Crystals ◽

Fat Body ◽

Competitive Inhibitor ◽

Dense Core ◽

Urate Oxidase ◽

Blood Proteins ◽

Free Base ◽

The Core ◽

The Matrix

The fat body of insects has always been compared functionally to the liver of vertebrates. Both synthesize and store glycogen and lipid and are concerned with the formation of blood proteins. The comparison becomes even more apt with the discovery of microbodies and the localization of urate oxidase and catalase in insect fat body.The microbodies are oval to spherical bodies about 1μ across with a depression and dense core on one side. The core is made of coiled tubules together with dense material close to the depressed membrane. The tubules may appear loose or densely packed but always intertwined like liquid crystals, never straight as in solid crystals (Fig. 1). When fat body is reacted with diaminobenzidine free base and H2O2 at pH 9.0 to determine the distribution of catalase, electron microscopy shows the enzyme in the matrix of the microbodies (Fig. 2). The reaction is abolished by 3-amino-1, 2, 4-triazole, a competitive inhibitor of catalase. The fat body is the only tissue which consistantly reacts positively for urate oxidase. The reaction product is sharply localized in granules of about the same size and distribution as the microbodies. The reaction is inhibited by 2, 6, 8-trichloropurine, a competitive inhibitor of urate oxidase.

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Electron Microscopy of Bacteriophage T7 Internal Head Proteins

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116814 ◽

1975 ◽

Vol 33 ◽

pp. 638-639

Author(s):

P. Serwer

Keyword(s):

Electron Microscopy ◽

Bacteriophage T7 ◽

Specimen Preparation ◽

Dna Molecule ◽

The Core ◽

Internal Core ◽

Phage T7 ◽

T7 Phage ◽

Preparation Techniques ◽

Internal Head

The genome of bacteriophage T7 is a duplex DNA molecule packaged in a space whose volume has been measured to be 2.2 x the volume of the B form of T7 DNA. To help determine the mechanism for packaging this DNA, the configuration of proteins inside the phage head has been investigated by electron microscopy. A core which is roughly cylindrical in outline has been observed inside the head of phage T7 using three different specimen preparation techniques.When T7 phage are treated with glutaraldehyde, DNA is ejected from the head often revealing an internal core (dark arrows in Fig. 1). When both the core and tail are present in a particle, the core appears to be coaxial with the tail. Core-tail complexes sometimes dislodge from their normal location and appear attached to the outside of a phage head (light arrow in Fig. 1).

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Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽

10.1093/genetics/149.1.45 ◽

1998 ◽

Vol 149 (1) ◽

pp. 45-56

Author(s):

Luther Davis ◽

JoAnne Engebrecht

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Heterologous Expression ◽

Protein Translation ◽

Developmental Pathways ◽

G1 Phase ◽

Growth Defect ◽

Wild Type ◽

Drosophila Homolog ◽

Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

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A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5130-5143.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5130-5143 ◽

Cited By ~ 32

Author(s):

Christine Soustelle ◽

Laurence Vernis ◽

Karine Fréon ◽

Anne Reynaud-Angelin ◽

Roland Chanet ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Nonhomologous End Joining ◽

End Joining ◽

Recombination Mechanism ◽

Growth Defects ◽

Protein Conjugates ◽

Synthetically Lethal ◽

Mutant Cells ◽

Insight Into

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

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CSE4 Genetically Interacts With the Saccharomyces cerevisiae Centromere DNA Elements CDE I and CDE II but Not CDE III: Implications for the Path of the Centromere DNA Around a Cse4p Variant Nucleosome

Genetics ◽

10.1093/genetics/156.3.973 ◽

2000 ◽

Vol 156 (3) ◽

pp. 973-981

Author(s):

Kevin C Keith ◽

Molly Fitzgerald-Hayes

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Loss ◽

Structural Studies ◽

Wild Type ◽

Histone Fold ◽

Histone Fold Domain ◽

Histone H3 Variant ◽

Dna Elements ◽

Mutant Cells ◽

Loss Rates

Abstract Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

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Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

Molecular and Cellular Biology ◽

10.1128/mcb.3.3.457-465.1983 ◽

1983 ◽

Vol 3 (3) ◽

pp. 457-465

Author(s):

C H Kim ◽

J R Warner

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Temperature Shift ◽

Restrictive Temperature ◽

Ribosomal Protein Genes ◽

Wild Type ◽

Temperature Shock ◽

Mild Temperature ◽

Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

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