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C W Akey

Publications and source records attributed to C W Akey.

At least 19 recordsLinked to original sources
PubMed2001

The crystal structure of nucleoplasmin-core: implications for histone binding and nucleosome assembly.

The efficient assembly of histone complexes and nucleosomes requires the participation of molecular chaperones. Currently, there is a paucity of data on their mechanism of action. We now present the structure of an N-terminal domain of nucleoplasmin (Np-core) at 2.3 A resolution. The Np-core monomer is an eight-stranded beta barrel that fits snugly within a stable pentamer. In the crystal, two pentamers associate to form a decamer. We show that both Np and Np-core are competent to assemble large complexes that contain the four core histones. Further experiments and modeling suggest that these complexes each contain five histone octamers which dock to a central Np decamer. This work has important ramifications for models of histone storage, sperm chromatin decondensation, and nucleosome assembly.

Amino Acid Sequence↗
PubMed2000

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments.

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data. Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.

Animals↗
PubMed2000

The structure of ribosome-channel complexes engaged in protein translocation.

Cotranslational translocation of proteins requires ribosome binding to the Sec61p channel at the endoplasmic reticulum (ER) membrane. We have used electron cryomicroscopy to determine the structures of ribosome-channel complexes in the absence or presence of translocating polypeptide chains. Surprisingly, the structures are similar and contain 3-4 connections between the ribosome and channel that leave a lateral opening into the cytosol. Therefore, the ribosome-channel junction may allow the direct transfer of polypeptides into the channel and provide a path for the egress of some nascent chains into the cytosol. Moreover, complexes solubilized from mammalian ER membranes contain an additional membrane protein that has a large, lumenal protrusion and is intercalated into the wall of the Sec61p channel. Thus, the native channel contains a component that is not essential for translocation.

Animals↗
PubMed1999

The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex.

The SecYEG complex is a major component of the protein translocation apparatus in the cytoplasmic membrane of bacteria. We have purified a translocationally active complex of the two subunits, SecY and SecE, from Bacillus subtilis. As demonstrated by electron microscopy, SecY/E forms ring structures in detergent solution and in intact lipid bilayers, often with a quasi-pentagonal appearance in projection. The particles represent oligomeric assemblies of the SecY/E complex and are similar to those formed by the eukaryotic Sec61p complex. We propose that these SecY/E rings represent protein-conducting channels and that the two essential membrane components SecY and SecE are sufficient for their formation.

Bacillus subtilis↗
PubMed1998

Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.

We have calculated a three-dimensional map of the yeast nuclear pore complex (yNPC) from frozen-hydrated specimens, thereby providing a direct comparison with the vertebrate NPC. Overall, the smaller yNPC is comprised of an octagonal inner spoke ring that is anchored within the nuclear envelope by a novel membrane-interacting ring. In addition, a cylindrical transporter is located centrally within the spokes and exhibits a variable radial expansion in projection that may reflect gating. The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they are required to form a functional NPC. However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences in nuclear transport regulation or mitotic behavior.

Animals↗
PubMed1998

Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export.

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

Animals↗
PubMed1997

The yeast spindle pole body is assembled around a central crystal of Spc42p.

The spindle pole body (SPB) is the microtubule organizing center (MTOC) in the yeast Saccharomyces that plays a pivotal role in such diverse processes as mitosis, budding, and mating. We have used cryoelectron microscopy and image processing to study the structure of isolated diploid SPBs. We show that SPBs are present in two lateral-size classes, sharing a similar vertical architecture comprised of six major layers. Tomographic reconstructions of heparin-stripped SPBs reveal a central hexagonally packed layer. Overexpression of Spc42p results in the growth of a similar layer, forming a crystal that encircles the SPB. Hence, the SPB is an MTOC that utilizes crystallographic packing of subunits in its construction.

Centrosome↗
PubMed1996

Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation.

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Animals↗
PubMed1995

Structural plasticity of the nuclear pore complex.

The nuclear pore complex (NPC) is strategically located at continuous junctions of the inner and outer nuclear membranes to catalyze macromolecular transport, without impending the diffusion of small molecules. In this paper, the structural plasticity of 4412 NPCs in isolated nuclear envelopes has been evaluated, utilizing correspondence analysis, classification and difference mapping. The data are grouped into seven clusters comprising two major groups, based on the degree of radial compaction within spokes and the symmetry of the inner spoke ring. The results have been correlated with differences in spoke domain packing observed in two published three-dimensional maps suggesting that symmetrical detergent-extracted NPCs are similar, but not identical to the most probable in vivo structure. A model is proposed in which spoke architecture is responsive to changes in the turgor pressure of the nuclear envelope. For example, detergent extraction may allow the outward facing domains of each spoke to adopt a radially-extended configuration while osmotic swelling may induce an inwards displacement, resulting in a radially compact spoke. Difference maps between approximately 822 symmetric projections of NPCs in membranes and after detergent-extraction have localized the nuclear envelope border. The data place limits on the radial and circumferential dimensions of diffusion channels (approximately 0 to 20 A x 190 A), proposed to reside at the pore periphery. The results confirm the observation that each spoke penetrates the nuclear envelope, linking up with the radial arms to form a "lumenal ring". Finally, putative closed, open and in-transit forms of the transporter are found with the same relative frequency in membrane-associated NPCs with radially compact or extended spokes; hence spoke deformations in isolated envelopes may be induced by experimental factors. However, concerted movements of the spoke domains (if reversible) may be utilized in the biological function of the NPC and some examples are given.

Analysis of Variance↗
PubMed1993

Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.

The nuclear pore complex spans the nuclear envelope and functions as a macromolecular transporter in the ATP-dependent process of nucleocytoplasmic transport. In this report, we present three dimensional (3D) structures for both membrane-associated and detergent-extracted Xenopus NPCs, imaged in frozen buffers by cryo-electron microscopy. A comparison of the differing configurations present in the 3D maps suggests that the spokes may possess an intrinsic conformational flexibility. When combined with recent data from a 3D map of negatively stained NPCs (Hinshaw, J. E., B. O. Carragher, and R. A. Milligan. 1992. Cell. 69:1133-1141), these observations suggest a minimal domain model for the spoke-ring complex which may account for the observed plasticity of this assembly. Moreover, lumenal domains in adjacent spokes are interconnected by radial arm dimers, forming a lumenal ring that may be responsible for anchoring the NPC within the nuclear envelope pore. Importantly, the NPC transporter is visualized as a centrally tapered cylinder that spans the entire width of the NPC, in a direction normal to the nuclear envelope. The central positioning, tripartite structure, and hollow nature of the transporter suggests that it may form a macromolecular transport channel, with a globular gating domain at each end. Finally, the packing of the transporter within the spokes creates a set of eight internal channels that may be responsible, in part, for the diffusion of ions and small molecules across the nuclear envelope.

Animals↗
PubMed1991

Probing the structure and function of the nuclear pore complex.

Nucleocytoplasmic transport is a bi-directional process mediated by the nuclear pore complex (NPC), which results in a segregation of cytoplasmic and nuclear macromolecules within cells. Some progress has been made in understanding the mechanistic basis of this selective transport phenomenon. In particular, cryo-electron microscopy of frozen-hydrated nuclear envelopes coupled with image processing and labeling studies, has provided a glimpse of the transporter at the center of the NPC.

Amino Acid Sequence↗
PubMed1990

Visualization of transport-related configurations of the nuclear pore transporter.

The transport of macromolecules between the cytoplasm and nucleus of the cell is mediated by the nuclear pore complex (NPC). In this study, details of the central transporter assembly within NPCs have been examined by cryoelectron microscopy, image processing, and classification analysis. The NPC transporter in isolated amphibian nuclei appears to adopt a minimum of four transport-related configurations including: (a) a putative closed form with a 90-100 A diameter central pore, (b) a docked form with material aligned over the pore, (c) an open form with substrates apparently caught "in transit," and (d) an open form with an enlarged pore. This data confirms previous observations on NPC transporters labeled with nucleoplasmin-gold (Akey, C.W., and D.S. Goldfarb. 1989. J. Cell Biol. 109:971-982) and allows a working model of the central NPC transporter to be proposed. The model is comprised of two supramolecular irislike assemblies which open asynchronously to provide an expanded pore for translocation while maintaining transport fidelity.

Animals↗
PubMed1989

Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

Animals↗
PubMed1989

Protein import through the nuclear pore complex is a multistep process.

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.

Acetylglucosamine↗
PubMed1987

The innermost chorionic layer of Drosophila. I. The role of chorin octamers in the formation of a family of interdigitating crystalline plates.

The innermost chorionic layer (ICL) within egg shells of Drosophila melanogaster is composed of thin, abutting three-dimensional crystalline plates which form a closed, membrane-like sheath. Collectively, the crystals within the sheath appear to form a family of related three-dimensional crystals in space group C222; however, specimens prepared for electron microscopy are actually two-dimensional crystals in c222. The projected structures of the negatively stained crystals have been studied by minimal dose electron microscopy employing image reconstruction methods. Thin sections indicate that unit cells within the ICL are composed of paired layers; top and bottom layers are related by centrally located 2-fold axes, aligned parallel to the surface of the ICL. The most probable structural unit of the crystals is a tetramer of chorin dimers with a point group symmetry of 222, which is denoted a chorin octamer. Projection maps were computed from average transforms of two-dimensional crystals for delta (the primitive unit cell angle) equal to 84 degrees, 90 degrees and 97 degrees (+/- 1.5 degrees). The maps indicate that the molecular transitions responsible for the observed family of crystals involve concerted intramolecular rearrangements about molecular 2-fold axes. The significance in vivo of the family of crystals within the ICL is not known; however, structural considerations suggest that the observed polymorphism may reflect one facet of an intrinsic bonding flexibility of the ICL octamer that may play a role in the formation of interplate junctions and the assembly of a continuous closed sheath. The ICL may therefore serve as a structural bridge between the vitelline membrane-wax layer and the endochondrial floor, allowing the larva to shed the inner egg shell layers during hatching.

Animals↗
PubMed1987

The innermost chorionic layer of Drosophila. II. Three-dimensional structure determination of the 90 degrees crystal form by electron microscopy.

The innermost chorionic layer (ICL) within egg shells of Drosophila is composed of a family of related, thin three-dimensional crystals that form a continuous sheath encapsulating the egg shell lumen. Junctions formed by interdigitating lattices play a central role in the construction of this macroscopic assembly. The three-dimensional structure of a two-dimensional crystal isolated from the ICL, with a primitive lattice angle delta of 90 degrees, has been determined from a complete tilt series of a negatively contrasted specimen at a resolution of 25 A. Inspection of the three-dimensional transform after data merging revealed that the space group is c222 and this symmetry was employed to generate a three-dimensional structure. The basic structural unit of the ICL is an octamer, described formally as a tetramer of dimers with point group symmetry 222. There are two classes of dimer in the octamer designated alpha and beta. The chorin octamer is composed of two classes of bent dimers, which make intramolecular contacts at the top and bottom of the molecule. The alpha-dimers are curved outwards away from the crystallographic 2-fold axis, while the beta-dimers are curved towards the molecular center. In addition, lattice contacts are formed primarily by beta-chorin dimers at both the top and bottom surfaces of the unit cell. The molecular weight of a chorin octamer determined from the analysis is about 6 X 10(5). The conformation of the chorin octamer determined here suggests that permutations of a basic molecular mechanism may be adequate to explain both the observed lattice polymorphisms of the ICL and the formation of interplate junctions necessary for the assembly of the macroscopic sheath.

Animals↗
PubMed1984

Trigonal catalase crystals: a new molecular packing assignment obtained from sections preserved with tannic acid.

The molecular packing of a trigonal crystal form of catalase initially studied by Longley [1] has been re-evaluated. Sections of crystals fixed and preserved with tannic acid were obtained parallel to the (001) and (100) planes. Specimens prepared by either conventional or low temperature embedding maintained 20 A resolution after sectioning. The space group of the crystals is either P3(1)21 or P3(2)21 and the observed unit cell parameters for (001) and (100) are a = b = 174 A, gamma = 119 degrees and b = 189 A, c = 248 A with alpha = 89.5 degrees. Computer-based reconstructions of two principal projections coupled with crystal density measurements allowed the deduction that there is one catalase tetramer per asymmetric unit. The crystal structure consists of 6 molecules packed closely about a common triad screw axis. This interpretation differs from that proposed by Longley [J. Mol. Biol. 30 (1967) 323], because thin sections of embedded crystals were assumed a priori to be positively stained in the early work; in actuality the sections were negatively stained. We also demonstrate that tannic acid fixation can lead to well preserved, positively stained crystal sections under certain conditions.

Animals↗