ABSTRACT
Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase , Adaptor Proteins, Signal Transducing , Dynactin Complex , Dyneins , Microtubule-Associated Proteins , Nerve Tissue Proteins , Cryoelectron Microscopy , Dynactin Complex/chemistry , Dynactin Complex/genetics , Dynactin Complex/metabolism , Dyneins/chemistry , Dyneins/genetics , Dyneins/metabolism , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Protein Binding , Humans , HeLa Cells , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , WD40 Repeats , Protein Interaction MappingABSTRACT
Fatty acids (FAs) play a central metabolic role in living cells as constituents of membranes, cellular energy reserves, and second messenger precursors. A 2.6 MDa FA synthase (FAS), where the enzymatic reactions and structures are known, is responsible for FA biosynthesis in yeast. Essential in the yeast FAS catalytic cycle is the acyl carrier protein (ACP) that actively shuttles substrates, biosynthetic intermediates, and products from one active site to another. We resolve the S. cerevisiae FAS structure at 1.9 Å, elucidating cofactors and water networks involved in their recognition. Structural snapshots of ACP domains bound to various enzymatic domains allow the reconstruction of a full yeast FA biosynthesis cycle. The structural information suggests that each FAS functional unit could accommodate exogenous proteins to incorporate various enzymatic activities, and we show proof-of-concept experiments where ectopic proteins are used to modulate FAS product profiles.
Subject(s)
Acyl Carrier Protein , Fatty Acids , Saccharomyces cerevisiae , Acyl Carrier Protein/chemistry , Catalytic Domain , Fatty Acids/biosynthesis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Transcription Factors/metabolism , Active Transport, Cell Nucleus , CRISPR-Cas Systems , Cell Line, Tumor , Female , Genes, myc , Humans , Male , Mitosis , Proteasome Endopeptidase Complex/chemistry , Protein Binding , ProteolysisABSTRACT
Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.
Subject(s)
Fatty Acid Synthases/chemistry , Fatty Acid Synthases/metabolism , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/metabolism , Acyltransferases/metabolism , Binding Sites , Catalytic Domain , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , Fatty Acids/biosynthesis , Fatty Acids/chemistry , Models, Molecular , Protein Subunits/chemistry , Protein Subunits/isolation & purification , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Better knowledge of unexpected fetal loss is the promise for better parental counseling and for prevention of recurrences. Fetal autopsy can provide a clue to ascertain cause of death in these cases. Variations in the incidence can be attributed to multiple factors. The present study was carried out to help us to develop a database concerning number of autopsies, incidence and types of congenital malformations (CMF) in the North-Western Indian population. The period of study was from January 2010 to November 2011. Autopsy was carried out on 150 fetuses following guidelines provided by a fetal autopsy protocol. Prior to autopsy, prenatal investigations such as ultrasound and radiographs were procured; a brief maternal and family history was noted. Out of a total of 150 autopsies, 87(58%) were induced abortions and 63(42%) spontaneous abortions. In total, the incidence of CMF was 104(69%) of fetal autopsies. The types of CMF were classified as central nervous system defects (CNS) in 49 (33%), gastrointestinal tract (GIT) disorders in 48 (32%), musuculoskeletal (MS) disorders in 31 (21%), genito-urinary (GU) in 25 (17%), and genetic disorders in 12 (8%). Multiple anomalies were present in 40 (27%) fetuses. Anencephaly (meroencephaly) turned out to be the most prevalent anomaly (29%). A few cases showed the occurrence of some uncommon syndromes. Major CMFs manifested very early in intra-uterine life, and could lead to termination of pregnancy (spontaneous or induced) in the 2nd trimester of gestation. Hence the presence of any CMF at the time of birth cannot provide the total percentage of CMF occurring in a given population. The above findings are discussed in the light of the available literature (AU)
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