Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex [preprint]

Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC

Structure of the SecY channel during initiation of protein translocation

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel

Electromagnetic Reduction of Plasma Density During Atmospheric Reentry and Hypersonic Flights

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/76341/1/AIAA-32147-259.pd

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of diseas

Genetic effects on gene expression across human tissues

Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression levels across individuals and diverse tissues of the human body, many of which are not easily accessible. Here we describe genetic effects on gene expression levels across 44 human tissues. We find that local genetic variation affects gene expression levels for the majority of genes, and we further identify inter-chromosomal genetic effects for 93 genes and 112 loci. On the basis of the identified genetic effects, we characterize patterns of tissue specificity, compare local and distal effects, and evaluate the functional properties of the genetic effects. We also demonstrate that multi-tissue, multi-individual data can be used to identify genes and pathways affected by human disease-associated variation, enabling a mechanistic interpretation of gene regulation and the genetic basis of disease

Apoptosome Structure, Assembly, and Procaspase Activation

Apaf-1-like molecules assemble into a ring-like platform known as the apoptosome. This cell death platform then activates procaspases in the intrinsic cell death pathway. In this review, crystal structures of Apaf-1 monomers and CED-4 dimers have been combined with apoptosome structures to provide insights into the assembly of cell death platforms in humans, nematodes, and flies. In humans, the caspase recognition domains (CARDs) of procaspase-9 and Apaf-1 interact with each other to form a CARD-CARD disk, which interacts with the platform to create an asymmetric proteolysis machine. The disk tethers multiple pc-9 catalytic domains to the platform to raise their local concentration, and this leads to zymogen activation. These findings have now set the stage for further studies of this critical activation process on the apoptosome

Oxidized or Reduced Cytochrome <i>c</i> and Axial Ligand Variants All Form the Apoptosome <i>in Vitro</i>

Cytochrome <i>c</i> (cyt <i>c</i>) has two important roles in vertebrates: mitochondrial electron transport and activating the intrinsic cell death pathway (apoptosis). To initiate cell death, cyt <i>c</i> dissociates from the inner mitochondrial membrane and migrates to the cytosol. In the cytosol, cyt <i>c</i> interacts stoichiometrically with apoptotic protease activating factor 1 (Apaf-1) and upon ATP binding induces formation of the heptameric apoptosome. It is not clear however what the redox state of cyt <i>c</i> is when it functions as the “active signal” for apoptosis. Some reports have indicated that only ferri (i.e., oxidized Fe³⁺ heme) but not ferro (reduced, Fe²⁺ heme) cyt <i>c</i> forms the apoptosome. Facilitated by our recently described recombinant system for synthesizing novel human cyt <i>c</i> proteins, we use a panel of cyt <i>c</i> axial ligand variants that exhibit a broad range of redox potentials. These variants exist in different redox states. Here we show that cyt <i>c</i> wild type and cyt <i>c</i> H19M (reduced state) and cyt <i>c</i> M81A and cyt <i>c</i> M81H (oxidized state) all bind to Apaf-1 and form the apoptosome

Structural insight into the protein translocation channel

A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time