The NuA4/TIP60 acetyltransferase complex is required for gene regulation, DNA repair and cell cycle progression. The limited structural information impeded understanding of NuA4/TIP60 assembly and ...regulatory mechanism. Here, we report the 4.7 Å cryo-electron microscopy (cryo-EM) structure of a NuA4/TIP60 TEEAA assembly (Tra1, Eaf1, Eaf5, actin and Arp4) and the 7.6 Å cryo-EM structure of a TEEAA-piccolo assembly (Esa1, Epl1, Yng2 and Eaf6). The Tra1 and Eaf1 constitute the assembly scaffold. The Eaf1 SANT domain tightly binds to the LBE and FATC domains of Tra1 by ionic interactions. The actin/Arp4 peripherally associates with Eaf1 HSA domain. The Eaf5/7/3 (TINTIN) and piccolo modules largely pack against the FAT and HEAT repeats of Tra1 and their association depends on Eaf1 N-terminal and HSA regions, respectively. These structures elucidate the detailed architecture and molecular interactions between NuA4 subunits and offer exciting insights into the scaffolding and regulatory mechanisms of Tra1 pseudokinase.
Macroautophagy (hereafter referred to as autophagy) degrades various intracellular constituents to regulate a wide range of cellular functions, and is also closely linked to several human diseases. ...In selective autophagy, receptor proteins recognize degradation targets and direct their sequestration by double-membrane vesicles called autophagosomes, which transport them into lysosomes or vacuoles. Although recent studies have shown that selective autophagy is involved in quality/quantity control of some organelles, including mitochondria and peroxisomes, it remains unclear how extensively it contributes to cellular organelle homeostasis. Here we describe selective autophagy of the endoplasmic reticulum (ER) and nucleus in the yeast Saccharomyces cerevisiae. We identify two novel proteins, Atg39 and Atg40, as receptors specific to these pathways. Atg39 localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of part of the nucleus. Atg40 is enriched in the cortical and cytoplasmic ER, and loads these ER subdomains into autophagosomes. Atg39-dependent autophagy of the perinuclear ER/nucleus is required for cell survival under nitrogen-deprivation conditions. Atg40 is probably the functional counterpart of FAM134B, an autophagy receptor for the ER in mammals that has been implicated in sensory neuropathy. Our results provide fundamental insight into the pathophysiological roles and mechanisms of 'ER-phagy' and 'nucleophagy' in other organisms.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell ...cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4-Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane ...(IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed ...repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.
Target identification is an essential basis for novel-pesticide development in new molecular design and lead optimization. ...3-(4-Methyl-1,2,3-thiadiazolyl)-6-trichloromethyl1,2,4triazolo3,4-b1,3,4thiadizole (YZK-C22) is a novel fungicide candidate with specific antifungal activity. We investigated its mode of action, and our studies indicated that YZK-C22 showed no cross resistance against Saccharomyces cerevisiae mutants with classic fungicide targets. Mec1 and Rad53 are two kinases that respond to DNA-replication damage, and the efficacy test showed that YZK-C22 could not perform its fungicidal activity by inhibiting DNA repair. Target screening by drug-affinity-responsive target stability (DARTS) showed that pyruvate kinase (PK), a key enzyme in the glycolytic pathway, was the potent new fungicidal target of YZK-C22. Fifty-eight differentially expressed proteins (DEPs) primarily involved in the metabolic process were identified by isobaric tags for relative and absolute quantification analysis (iTRAQ) in S. cerevisiae, and protein expression in the citrate cycle decreased with treatment of 5 mg/L YZK-C22, which was consistent with the results of DARTS. Molecular-docking analysis further validated that YZK-C22 could dock into the active center of PK instead of phosphoenolpyruvate. The enzyme activity of PK from S. cerevisiae was competitively inhibited with a K i of 3.33 ± 0.28 μmol/L, and the cell-growth inhibition of S. cerevisiae was released by supplementation with pyruvic acid, whereas the growth of S. cerevisiae was not recovered by adding PK’s substrate (phosphoenolpyruvate) or allosteric regulator (fructose-1,6-bisphosphate). The present studies uncovered and validated the primary target of the new, potent fungicidal candidate YZK-C22; our results provide a successful, valuable, and applicable case of target discovery and identification for novel-fungicide development.
Many cells are remarkably proficient at tracking very shallow chemical gradients, despite considerable noise from stochastic receptor-ligand interactions. Motile cells appear to undergo a biased ...random walk: spatial noise in receptor activity may determine the instantaneous direction, but because noise is spatially unbiased, it is filtered out by time averaging, resulting in net movement upgradient. How nonmotile cells might filter out noise is unknown.
Using yeast chemotropic mating as a model, we demonstrate that a polarized patch of polarity regulators “wanders” along the cortex during gradient tracking. Computational and experimental findings suggest that actin-directed membrane traffic contributes to wandering by diluting local polarity factors. The pheromone gradient appears to bias wandering via interactions between receptor-activated Gβγ and polarity regulators. Artificially blocking patch wandering impairs gradient tracking.
We suggest that the polarity patch undergoes an intracellular biased random walk that enables noise filtering by time averaging, allowing nonmotile cells to track shallow gradients.
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► A wandering polarity site distributes growth in yeast cells responding to pheromone ► Wandering, but not polarity establishment, is actin dependent ► A mathematical model incorporating polarity and vesicle traffic exhibits wandering ► Blocking wandering impairs gradient tracking
To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process ...involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cell size fundamentally affects all biosynthetic processes by determining the scale of organelles and influencing surface transport. Although extensive studies have identified many mutations ...affecting cell size, the molecular mechanisms underlying size control have remained elusive. In the budding yeast Saccharomyces cerevisiae, size control occurs in G1 phase before Start, the point of irreversible commitment to cell division. It was previously thought that activity of the G1 cyclin Cln3 increased with cell size to trigger Start by initiating the inhibition of the transcriptional inhibitor Whi5 (refs 6-8). Here we show that although Cln3 concentration does modulate the rate at which cells pass Start, its synthesis increases in proportion to cell size so that its total concentration is nearly constant during pre-Start G1. Rather than increasing Cln3 activity, we identify decreasing Whi5 activity--due to the dilution of Whi5 by cell growth--as a molecular mechanism through which cell size controls proliferation. Whi5 is synthesized in S/G2/M phases of the cell cycle in a largely size-independent manner. This results in smaller daughter cells being born with higher Whi5 concentrations that extend their pre-Start G1 phase. Thus, at its most fundamental level, size control in budding yeast results from the differential scaling of Cln3 and Whi5 synthesis rates with cell size. More generally, our work shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and ...toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from Saccharomyces cerevisiae solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo–electron microscopy structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions aremediated by conserved, pore-loop tyrosines that contact an 80-angstrom-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore loop–substrate interactions by two amino acids. These changes are coupled to activation of specific nucleotide hydrolysis sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and substrate-responsive flexibility during Hsp104-catalyzed disaggregation.
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