Annual Review of Biochemistry - Volume 76, 2007

Although cyclic nucleotide phosphodiesterases (PDEs) were described soon after the discovery of cAMP, their complexity and functions in signaling is only recently beginning to become fully realized. We now know that at least 100 different PDE proteins degrade cAMP and cGMP in eukaryotes. A complex PDE gene organization and a large number of PDE splicing variants serve to fine-tune cyclic nucleotide signals and contribute to specificity in signaling. Here we review some of the major concepts related to our understanding of PDE function and regulation including: (a) the structure of catalytic and regulatory domains and arrangement in holoenzymes; (b) PDE integration into signaling complexes; (c) the nature and function of negative and positive feedback circuits that have been conserved in PDEs from prokaryotes to human; (d) the emerging association of mutant PDE alleles with inherited diseases; and (e) the role of PDEs in generating subcellular signaling compartments.

Integration of multiple signaling pathways at the level of their transcriptional effectors provides an important strategy for fine-tuning gene expression and ensuring a proper program of development. Posttranslational modifications, such as phosphorylation, play important roles in modulating transcription factor activity. The discovery that the transcription factor Eyes absent (Eya) possesses protein phosphatase activity provides an interesting new paradigm. Eya may regulate the phosphorylation state of either itself or its transcriptional cofactors, thereby directly affecting transcriptional output. The identification of a growing number of transcription factors with enzymic activity suggests that such dual-function proteins exert greater control of signaling events than previously imagined. Given the conservation of both its phosphatase and transcription factor activity across mammalian species, Eya provides an excellent model for studying how a single protein integrates these two functions under the influence of multiple signaling pathways to promote development.

The positioning of a cytoskeletal element that dictates the division plane is a fundamental problem in biology. The assembly and positioning of this cytoskeletal element has to be coordinated with DNA segregation and cell growth to ensure that equal-sized progeny cells are produced, each with a copy of the chromosome. In most prokaryotes, cytokinesis involves positioning a Z ring assembled from FtsZ, the ancestral homologue of tubulin. The position of the Z ring is determined by a gradient of negative regulators of Z-ring assembly. In Escherichia coli, the Min system consists of three proteins that cooperate to position the Z ring through a fascinating oscillation, which inhibits the formation of the Z ring away from midcell. Additional gradients of negative regulators of FtsZ assembly are used by E. coli and other bacteria to achieve spatial control of Z-ring assembly.

The kinetochore is a key cell division organelle that enables high-fidelity transmission of genetic information by coupling chromosomes to spindle microtubules during mitosis and meiosis. Despite its cytological description more than a century ago, remarkably little information is available on kinetochore function at a molecular level. Recently, important advances elucidating the overall organization of kinetochores, as well as information about the structures and molecular mechanisms of kinetochore function, have been achieved through a detailed analysis of the kinetochores of the budding yeast Saccharomyces cerevisiae. Here we review the current understanding of kinetochore function in budding yeast and draw comparisons to recent findings in other organisms.

Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.

Remarkable strides have been made over the past 20 years in elucidating the molecular basis of membrane trafficking (1). Indeed, a combination of biochemical and genetic approaches have determined the identity and function of many of the core constituents needed for protein secretion and endocytosis. But much remains to be learned. This review highlights underlying themes in membrane traffic to help us refocus and solve many remaining and newly emerging issues that are fundamental to mammalian cell biology and human physiology.

In eukaryotic cells, segregation of DNA replication and RNA biogenesis in the nucleus and protein synthesis in the cytoplasm poses the requirement of transporting thousands of macromolecules between the two cellular compartments. Transport between nucleus and cytoplasm is mediated by soluble receptors that recognize specific cargoes and carry them through the nuclear pore complex (NPC), the sole gateway between the two compartments at interphase. Nucleocytoplasmic transport is specific not only in terms of cargo recognition, but also in terms of directionality, with nuclear proteins imported into the nucleus and RNAs exported from it. How is directionality achieved? How can the receptors be both specific and versatile in recognizing a multitude of cargoes? And how can their interaction with NPCs allow fast translocation? We describe the molecular mechanisms underlying nucleocytoplasmic transport as they have been revealed by structural studies of the receptors and regulators in different steps of transport cycles.

For more than a century after their discovery, mitochondria were viewed almost exclusively as ATP-generating metabolic units. This view changed with the discovery of the mitochondrial genetic system and the central role of mitochondria in programmed cell death. As a result, much of the current research focuses on the complex interplay between mitochondria and the rest of the eukaryotic cell. This interplay is central to mitochondrial biogenesis. The following five reviews published in this volume summarize recent discoveries in this area: DNA Replication and Transcription in Mammalian Mitochondria, Mitochondrial-Nuclear Communications, Translocation of Proteins into Mitochondria, The Machines that Divide and Fuse Mitochondria, and Why Do We Still Have a Maternally Inherited Mitochondrial DNA? Insights from Evolutionary Medicine.

The mitochondrion was originally a free-living prokaryotic organism, which explains the presence of a compact mammalian mitochondrial DNA (mtDNA) in contempory mammalian cells. The genome encodes for key subunits of the electron transport chain and RNA components needed for mitochondrial translation. Nuclear genes encode the enzyme systems responsible for mtDNA replication and transcription. Several of the key components of these systems are related to proteins replicating and transcribing DNA in bacteriophages. This observation has led to the proposition that some genes required for DNA replication and transcription were acquired together from a phage early in the evolution of the eukaryotic cell, already at the time of the mitochondrial endosymbiosis. Recent years have seen a rapid development in our molecular understanding of these machineries, but many aspects still remain unknown.

Mitochondria cannot be made de novo but replicate by a mechanism of recruitment of new proteins, which are added to preexisting subcompartments. Although mitochondria have their own DNA, more than 98% of the total protein complement of the organelle is encoded by the nuclear genome. Mitochondrial biogenesis requires a coordination of expression of two genomes and therefore cross talk between the nucleus and mitochondria. In mammals, regulation of mitochondrial biogenesis and proliferation is influenced by external factors, such as nutrients, hormones, temperature, exercise, hypoxia, and aging. This complexity points to the existence of a coordinated and tightly regulated network connecting different pathways. Communications are also required for eliciting mitochondrial responses to specific stress pathways. This review covers the mechanisms of mitochondrial biogenesis and the way cells respond to external signals to maintain mitochondrial function and cellular homeostasis.

About 10% to 15% of the nuclear genes of eukaryotic organisms encode mitochondrial proteins. These proteins are synthesized in the cytosol and recognized by receptors on the surface of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intramitochondrial sorting of these proteins; ATP and the membrane potential are used as energy sources. Chaperones and auxilliary factors assist in the folding and assembly of mitochondrial proteins into their native, three-dimensional structures. This review summarizes the present knowledge on the import and sorting of mitochondrial precursor proteins, with a special emphasis on unresolved questions and topics of current research.

Mitochondria are derived from eubacteria; however, in most eukaryotes, novel mechanisms for the propagation of this organelle and its genome have evolved. This review focuses on what is currently known about the novel molecular machines that divide and fuse mitochondria.

The human cell is a symbiosis of two life forms, the nucleus-cytosol and the mitochondrion. The nucleus-cytosol emphasizes structure and its genes are Mendelian, whereas the mitochondrion specializes in energy and its mitochondrial DNA (mtDNA) genes are maternal. Mitochondria oxidize calories via oxidative phosphorylation (OXPHOS) to generate a mitochondrial inner membrane proton gradient (ΔP). ΔP then acts as a source of potential energy to produce ATP, generate heat, regulate reactive oxygen species (ROS), and control apoptosis, etc. Interspecific comparisons of mtDNAs have revealed that the mtDNA retains a core set of electron and proton carrier genes for the proton-translocating OXPHOS complexes I, III, IV, and V. Human mtDNA analysis has revealed these genes frequently contain region-specific adaptive polymorphisms. Therefore, the mtDNA with its energy controlling genes may have been retained to permit rapid adaptation to new environments.

Excitatory (glutamatergic) synapses in the mammalian brain are usually situated on dendritic spines, a postsynaptic microcompartment that also harbors organelles involved in protein synthesis, membrane trafficking, and calcium metabolism. The postsynaptic membrane contains a high concentration of glutamate receptors, associated signaling proteins, and cytoskeletal elements, all assembled by a variety of scaffold proteins into an organized structure called the postsynaptic density (PSD). A complex machine made of hundreds of distinct proteins, the PSD dynamically changes its structure and composition during development and in response to synaptic activity. The molecular size of the PSD and the stoichiometry of many major constituents have been recently measured. The structures of some intact PSD proteins, as well as the spatial arrangement of several proteins within the PSD, have been determined at low resolution by electron microscopy. On the basis of such studies, a more quantitative and geometrically realistic view of PSD architecture is emerging.