Showing papers in "Essays in Biochemistry in 2000"

Pore relations: nuclear pore complexes and nucleocytoplasmic exchange.

Abstract: NPCs are the sole sites of exchange between the nucleus and cytoplasm. A large family of transport factors carry cargo between the nucleus and cytoplasm through the NPC. The NPC is a huge symmetric octagonal structure comprised of dozens of NUPs. NUPs make many contacts with surrounding structures, including the NE, the cytoplasm and nuclear interior. A subset of NUPs contain repeated peptide motifs that serve as docking sites for transport factors. The directionality of transport is determined by the transport factor, and its interactions with the small GTPase Ran and NUPs. Very little is known about how the NPC mediates transport, NPC assembly and the NPC's role in regulating transport, but these areas of research are beginning to emerge.

F1-ATPase: a highly efficient rotary ATP machine.

Abstract: A single molecule of F1-ATPase is by itself a rotary motor in which a central subunit, gamma, rotates against a surrounding stator cylinder made of alpha 3 beta 3 hexamer. Driven by the three beta subunits that hydrolyse ATP sequentially, the motor runs with discrete 120 degrees steps at low ATP concentrations. Over broad ranges of load and speed, the motor produces a constant torque of 40 pN.nm. The mechanical work the motor does in the 120 degrees step, or the work per ATP hydrolysed, is also constant and amounts to 80-90 pN.nm, which is close to the free energy of ATP hydrolysis. Thus this motor can work at near 100% efficiency.

Glycosylation and protein transport.

Abstract: Transport along the secretory pathway is largely signal-mediated. Proteins in the secretory pathway can be covalently modified with various carbohydrate structures, most commonly O-glycans, N-glycans and/or proteoglycans. Carbohydrate modifications can change the physical properties of proteins or can function as specific recognition epitopes. Glycosylation can act as an apical sorting signal in polarized epithelial cells and provide a signal for surface transport in non-polarized fibroblasts. Homologues of leguminous plant lectins have been identified in yeast, fruitflies, worms and humans. Intracellular lectins are candidate receptors in the secretory pathway to mediate concentration of cargo in carrier vesicles.

RNA export: insights from viral models.

Abstract: The retroviruses export intron-containing RNA. The complex retroviruses encode a Rev protein that uses a leucine-rich NES to interact with CRM1 and the U snRNA-export pathway. Other viruses encode proteins with a Rev-like NES. The type-D retroviruses contain a CTE that binds the cellular protein TAP to export intron-containing RNA through the mRNA pathway. Intronless viral transcripts contain post-transcriptionally acting RNA elements that may compensate for the lack of an intron. The functions of elements in intronless RNA are not fully understood but may be in export and/or 3'-end processing.

Protein targeting and translocation at the endoplasmic reticulum membrane--through the eye of a needle?

Abstract: SRP-dependent and SRP-independent targeting routes deliver precursor proteins to the ER membrane translocon. These precursors are translocated into (for membrane proteins) and across (for secretory protein) the ER membrane via aqueous channels composed of oligomers of the Sec61 complex. Both ends of the ER translocon are 'gated' and the opening and closing of these gates are closely regulated. The lateral exit of hydrophobic polypeptide regions into the phospholipid bilayer also appears to be a carefully controlled process. Accessory components are transiently associated with active ER translocation sites and modify the nascent polypeptide as it appears on the luminal side of the membrane.

The molecular anatomy of dynein.

Abstract: Recent molecular, genetic and functional studies have led to an unparalleled growth in our understanding of dynein and the roles played by the various polypeptides of these massive macromolecular assemblies. Dyneins are highly complex 1-2MDa complexes that function as molecular motor and move the cargo to which they are attached towards the minus-end of a microtubule. Dynein motor function is a property of the heavy chains, whereas the intermediate chains are involved in attachment to the appropriate cargo. In order for useful work to be obtained, motor and cargo-binding activities must be tightly controlled. Current data suggest that this is the role played by certain accessory light-chain proteins. The LC8 is highly conserved and found in many enzyme systems. This protein is essential in multicellular organisms. The dynein light chains Tctex1 and Tctex2 have been implicated in the non-Mendelian transmission of variant forms of mouse chromosome 17.

The control of gene expression by regulated nuclear transport.

Abstract: Many proteins show distinct nuclear- and cytoplasmic-localization patterns. For proteins above the diffusion limit of the NPC, this localization is governed by the activity of NLSs and/or NESs contained in the protein. Structural modification of proteins can affect NLS and NES activities. Ligand binding, phosphorylation and proteolysis are each capable of modifying the nucleocytoplasmic distribution of proteins. In the case of transcription factors, control of these structural modifications affects access of the transcription factor to the chromatin. This management of cellular distribution, in turn, regulates gene expression.

The roles of unconventional myosins in hearing and deafness.

Abstract: The proper expression and function of several unconventional myosins are necessary for inner-ear function. Mutations in MYO7A and MYO15 cause deafness in humans, and mice. Whereas mutations in Myo6 cause inner-ear abnormalities in mice, as yet no human deafness has been found to the result of mutations in MYO6. In the mammalian inner ear there are at least nine different unconventional myosin isozymes expressed. Myosin 1 beta, VI, VIIa and probably XV are all expressed within a single cell in the inner ear, the hair cell. The myosin isozymes expressed in the hair cell all have unique domains of expression and in some areas, such as the pericuticular necklace, several domains overlap. This suggests that these myosins all have unique functions and that all are individually targeted within the hair cell. The mouse is proving to be a useful model organism for studying both human deafness and elucidating the normal functions of unconventional myosins in vivo.

Translational elongation factor G: a GTP-driven motor of the ribosome.

Abstract: EF-G is a large, five-domain GTPase that promotes the directional movement of mRNA and tRNAs on the ribosome in a GTP-dependent manner. Unlike other GTPases, but by analogy to the myosin motor, EF-G performs its function of powering translocation in the GDP-bound form; that is, in a kinetically stable ribosome-EF-G(GDP) complex formed by GTP hydrolysis on the ribosome. The complex undergoes an extensive structural rearrangement, in particular affecting the small ribosomal subunit, which leads to mRNA-tRNA movement. Domain 4, which extends from the 'body' of the EF-G molecule much like a lever arm, appears to be essential for the structural transition to take place. In a hypothetical model, GTP hydrolysis induces a conformational change in the G domain of EF-G which affects the interactions with neighbouring domains within EF-G. The resulting rearrangement of the domains relative to each other generates conformational strain in the ribosome to which EF-G is fixed. Because of structural features of the tRNA-ribosome complex, this conformational strain results in directional tRNA-mRNA movement. The functional parallels between EF-G and motor proteins suggest that EF-G differs from classical G-proteins in that it functions as a force-generating mechanochemical device rather than a conformational switch. There are other multi-domain GTPases that may function in a similar way.

Mechanisms of mitochondrial protein import.

Abstract: Mitochondria import most of their proteins from the cytosol. Precursor forms of most matrix proteins as well as some IM and IMS proteins are synthesized on cytoplasmic ribosomes with N-terminal cleavable signal sequences. Many other mitochondrial proteins including IM carrier proteins contain internal targeting sequences. Three multisubunit translocases, one in the OM and two in the IM, participate in the import process. These translocases co-operate with cytosolic chaperones, chaperone-like soluble proteins in the IMS as well as chaperones in the matrix. Insertion of carrier proteins into the IM only requires a membrane potential. On the other hand, translocation of preproteins across the IM into the matrix requires (i) a membrane potential, (ii) GTP hydrolysis, which occurs at the outer side of the IM, and (iii) ATP-dependent interactions occurring at the matrix side. Following import, the cleavable signal sequence of most preproteins is removed in one step by the MPP. In some cases, removal of the signal sequence is achieved in two steps; first by MPP and second by either mitochondrial intermediate peptidase or by IM peptidases. Imported proteins must be folded properly to perform their functions.

The molecular motors of cilia and eukaryotic flagella.

Abstract: Axonemal dyneins occur in two rows (as inner and outer arms) on each of the nine doublets. Axonemal dynein binds reversibly to the B-microtubule and has an ATP-insensitive anchorage to the A-microtubule of the adjacent doublet. The heavy chains have the form of globular heads and are responsible for chemo-mechanical transduction. The B-tubule-binding site is on a tenuous extension of the head. There is only one type of ODA. A 12 nm shift in the globular heads is associated with the hydrolysis cycle. There are three types of IDA. No functional changes have been recognized in their complex conformation. There is plentiful evidence that the axonemal dyneins produce interdoublet displacement. Doubt remains on how much sliding occurs per cycle of ATP hydrolysis. The mechanism for transforming sliding into bending is not yet explained.

The immunological properties of endoplasmic reticulum chaperones: a conflict of interest?

Abstract: ER chaperones are abundant and highly conserved proteins that display both peptide binding and chaperone activity. Of the family of chaperones present in the mammalian ER, GRP94 and calreticulin are apparently unique in their ability to elicit CD8+ T-cell responses against components of their bound-peptide pools. The ability of GRP94 and calreticulin to elicit CD8+ T-cell responses indicates that both proteins bind peptides suitable for assembly on to MHC class-I molecules. The capacity to function as molecular chaperones and as peptide-binding proteins capable of transferring, directly or indirectly, peptides on to class-I molecules, indicates that GRP94 and calreticulin participate in the regulation of both peptide and polypeptide traffic in the ER. Perspectives on the regulation of and interplay between the peptide binding and chaperone activity of GRP94 and calreticulin are discussed.

Microtubule-based transport along axons, dendrites and axonemes.

Abstract: MTs in cytoplasmic extensions including axons, dendrites and axonemes serve as polarized tracks for vectorial intracellular transport driven by MT-based motor proteins. Although axons and axonemes serve very different functions, increasing evidence suggests that the transport events, MT organization and the motors involved in their formation and function are conserved. Thus, there are obvious similarities in the mechanisms of axonal transport and IFT. The MT arrays of axons and axonemes are parallel, whereas those of dendrites are anti-parallel, but the functional significance of this difference and its consequences for mechanisms of transport along these processes are unclear. MT-based motor proteins of the dynein and kinesin superfamilies transport a variety of cargos including membrane-bound vesicles and macromolecular complexes along MTs of axons, dendrites and axonemes, and thus contribute to the formation, maintenance and function of these cytoplasmic extensions. Chemosensory neurons in the nematode C. elegans represent an appealing system for studying transport events along dendrites and axonemes that occur sequentially in a single cell.

Motor proteins of the kinesin superfamily: structure and mechanism

Abstract: Kinesins are ATP-driven microtubule motor proteins that produce directed force. The kinesin superfamily currently encompasses over 100 eukaryotic proteins containing a common motor domain. Both the nucleotide-binding fold and active-site chemistry of the motor domain are also present in the actin-based motor, myosin. Kinesins can be classified into three groups based on the position of their motor domains: N-terminal, C-terminal and internal kinesins. Conventional kinesin operates as a dimer, walking in a co-ordinated, hand-over-hand fashion along a microtubule protofilament. X-ray crystal structures and EM reconstructions show major differences in the quaternary arrangement of kinesin domains in minus-end- and plus-end-directed motors. Kinesin's neck region, directly adjacent to the motor domain, dictates directionality.

How to proteins move along DNA? Lessons from type-I and type-III restriction endonucleases.

Abstract: Protein-mediated communications on DNA are universally important. The translocation of DNA driven by a high-energy phosphoryl potential allows long stretches of DNA to be traversed without dissociation. Type-I and type-III enzymes both use a common DNA-tracking mechanism to move along DNA, dependent on the hydrolysis of ATP. Type-I enzymes cleave DNA at distant DNA sites (and in some cases close to the site), due to a stall in enzyme motion. This can be due to collision with another translocating type-I enzyme or, on circular DNA, due to an increased topological load. ATP hydrolysis is considerable, and continues after DNA cleavage. Type-III enzymes only cleave DNA proximal to their sites due to collision between two endonucleases tracking with defined polarity. ATP hydrolysis is less than with the type-I enzymes. Homology to DNA helicases has been found within the HsdR and Res subunits. Mutagenesis of the DEAD-box motifs affects both ATP hydrolysis and DNA cleavage. This demonstrates a tight link between ATPase and endonuclease activities. A strand-separation mechanism akin to the DNA helicases is a possibility. The DNA-based motor proteins are mechanistically ill-defined. Further study using some of the techniques pioneered with classical motor proteins will be needed to reveal more detail.

Nuclear transport: never-ending cycles of signals and receptors.

Abstract: Proteins transported into and out of the nucleus require amino acid motifs called NLSs and NESs, respectively. The amino acid sequences of these signals vary considerably. A superfamily of transport receptors has been identified and each member contains three transport-related domains. Transport receptors bind to the signal sequences, either directly or through adapter proteins, to promote nucleocytoplasmic transport. The diversity of signals, receptors and adapter proteins suggests that there are many pathways for nuclear entry or exit. The direction of transport (into or out of the nucleus) is regulated in part by the small GTPase Ran as well as by intrinsic substrate motifs.

Functions and origins of the chloroplast protein-import machinery.

Abstract: The vast majority of chloroplast proteins are nuclear-encoded and are imported into the organelle after synthesis in the cytoplasm. Targeting to chloroplasts is mediated by a variety of intrinsic targeting signals that direct the preprotein to its proper organelle subcompartment. Translocation at the envelope membrane is directed by the interactions of an N-terminal transit sequences on the preprotein and a general import machinery composed of the outer-membrane Toc machinery and the inner-membrane Tic machinery. The Toc and Tic components interact to bypass the intermembrane space and provide direct transport of preproteins from the cytoplasm to the stroma. There are at least four targeting pathways to the thylakoid membrane, the cpSec pathway, the delta pH pathway, the cpSRP pathway and the spontaneous pathway. These pathways require distinct intrinsic targeting signals, and apparently evolved to accommodate the translocation of classes of proteins with particular characteristics. Proteins similar to some components of the envelope and thylakoid translocation pathways are found in bacterial systems. However, a number of components do not have bacterial counterparts and are unique to the chloroplast pathways. It therefore appears that the chloroplast translocation systems have evolved from membrane-transport systems that were present in the original endosymbiont by incorporating proteins necessary to adapt to the constraints of endosymbiosis.

Muscle, myosin and single molecules.

Abstract: Whereas we have a great deal of information about myosin, there remain fundamental questions about its mechanism (and those of other motor proteins). Single-molecule technologies enable us to make measurements we cannot make from large ensembles of molecules. Optical tweezers (and similar techniques) are used to measure the mechanical aspects of actomyosin interactions, including force, displacement and stiffness. Single-molecule fluorescence has been used to observe the binding and release of nucleotide by myosins. A combination of these measurements has the potential to solve the problem of coupling of ATP hydrolysis to mechanical work in motor proteins.

Molecular motors in the heart.

Abstract: The contractile apparatus of muscle is a highly efficient and adaptable mechanism for producing movement and is exploited throughout the animal kingdom. Molecular biology is yielding important insights into the intricate functioning of muscular contraction, especially in the heart, and in explaining the genesis of inherited myopathies. Mutational analyses of the sarcomeric-protein genes in conjunction with clinical assessment have shown that certain mutations indicate a more serious prognosis in HCM. Detecting these mutations in individuals is important for screening other family members for the disease. Understanding the contractile apparatus at the molecular level could contribute to the design of more effective drugs for treatment of cardiac diseases.

Introduction: the mechano-biochemistry of molecular motors

Abstract: Molecular motors are mechano-enzymes, protein machines for transducing chemical potential energy into mechanical motion. Some general principles of their mechanisms are known from now-classical biochemical and physiological studies on muscle. The catalytic cleavage of a nucleotide (usually ATP) in the active site drives the motor into a metastable, mechanically strained conformation, and the subsequent relaxation of this conformation to an unstrained state is harnessed to do useful work. To gain more detailed information about structure–function relationships we need to tinker with each motor, much as a motor mechanic tinkers with a macroscopic engine. The questions are the same. What moves? Why does it move? What limits its performance? What happens when I pull this? Like the motor in your car, molecular motors have an operational cycle of forceful shape changes, and again like your car, this cycle is co-ordinated and driven by the chemistry of fuel consumption. For molecular-scale motors, it is the chemical catalytic cycle of ATP turnover in the motor active site that drives the development of mechanical strain. And just as the chemical transitions of ATP processing generate motion, so mechanical strain applied to the motor by an external load can feed back on the chemistry of the motor active site. This feedback allows the motor not only to generate force, but also to collaborate efficiently with other members of a team of motors. By sensing the forces produced by its team mates and adjusting its own force-generating schedule to fit in, a particular motor can contribute efficiently to a team effort. The prob-

Motors in muscle: the function of conventional myosin II.

Abstract: Solution measurements indicate that actin and myosin alternately bind and dissociate during one ATP hydrolysis cycle. Crystallographic studies indicate at least two basic conformations of the myosin head exist in which the regulatory domain swings through an angle of about 70 degrees. Actin must further modulate these conformations, but high-resolution information about the actomyosin interface is lacking. One-to-one coupling between the ATPase cycle and a mechanical cycle involving myosin-head bending could account for about a 10 nm stroke size. At high sliding velocities, discrepancies remain which suggest that a myosin head may undergo repetitive interactions with actin for each ATP hydrolysed.