How proteins enter the nucleus

Nuclear protein import is a selective process. Proteins destined for the nucleus contain NLSs. These short stretches of amino acids interact with proteins located in the cytoplasm, on the nuclear envelope, and/or at the nuclear pore complex. Following binding at the pore complex, proteins are translocated through the pore into the nucleus in a manner requiring ATP. The biochemical dissection of the nuclear pore complex has begun. Alteration of protein import into the nucleus is emerging as a new and complex form of regulation. However, we are left with the following problems: How do proteins move through the cytoplasm to reach the nuclear pore? How does the nuclear pore complex open and close in a selective manner? How is ATP utilized during import? And finally, how is bi-directional traffic of both proteins and RNA through the pore regulated?     

What are DNA sequence motifs?

Sequence motifs are becoming increasingly important in the analysis of gene regulation. How do we define sequence motifs, and why should we use sequence logos instead of consensus sequences to represent them? Do they have any relation with binding affinity? How do we search for new instances of a motif in this sea of DNA?     

Fifteen compelling open questions in plant cell biology

As scientists, we are at least as excited about the open questions—the things we do not know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such “rules” conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

We asked 15 experts to address what they consider to be the most compelling open questions in plant cell biology and these are their questions.     

Amphitropic proteins: regulation by reversible membrane interactions (review).

What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding.     

The modes of action of juvenile hormones: some questions we ought to ask

This paper argues that the current dogma that juvenile hormones are structurally unique and constitute a family of derivatives of farnesoic acid which are produced by the corpus allatum (CA), secreted into the hemolymph, frequently transported by binding proteins, enter cells by diffusion across the cell membrane and there the products of the CA interact in some way with the genome, probably via nuclear receptors of the steroid superfamily, may not be tenable. It does so by examining the following questions. How many JHs are there? Are there other sources of JH in insects? Are there non-farnesoids with JH activity in insects? How does JH get into cells? Is the product of the CA the effective hormone? How many modes of action are there? How many receptors are there?     

Similar binding sites and different partners: implications to shared proteins in cellular pathways

We studied a data set of structurally similar interfaces that bind to proteins with different binding-site structures and different functions. Our multipartner protein interface clusters enable us to address questions like: What makes a given site bind different proteins? How similar/different are the interactions? And, what drives the apparently less-specific association? We find that proteins with common binding-site motifs preferentially use conserved interactions at similar interface locations, despite the different partners. Helices are major vehicles for binding different partners, allowing alternate ways to achieve favorable association. The binding sites are characterized by imperfect packing, planar architectures, bridging water molecules, and, on average, smaller size. Interestingly, analysis of the connectivity of these proteins illustrates that they have more interactions with other proteins. These findings are important in predicting "date hubs," if we assume that "date hubs" are shared proteins with binding sites capable of transient binding to multipartners, linking higher-order networks.     

A budding and fusing journey through the secretory pathway. Genetic and In Vitro Analysis of Cell Compartmentalization: A UCLA symposium, Taos, NM, USA, February 3-9, 1990

Reconstitution of vesicular transport events and the molecular and genetic analysis of the secretory pathway have taken the field of membrane traffic into a new era. Already, proteins have been discovered that facilitate multiple transport steps, and studies of the identities and modes of action of additional transport components, such as those that specify the targets of transport vesicles, will soon follow. Even after we understand how transport vesicles form, find their targets, and then fuse, other fundamental questions will still remain. How are proteins sorted into distinct transport vesicles? How is the directionality of protein transport achieved? How do organelles maintain their identities in the face of large volumes of membrane traffic? Finally, how is membrane traffic regulated? Answers to each of these fundamental questions are likely to be available in the not-too-distant future.     

Protein-surfactant interactions: a tale of many states

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denature proteins, the field has kept a relatively low profile until the last decade. Within this period there has been a maturation of techniques for more accurate and sophisticated analyses of protein-surfactant complexes such as calorimetry and small angle scattering techniques. In this review I provide an overview of different useful approaches to study these complexes and identify eight different issues which define central concepts in the field. (1) Are proteins denatured by monomeric surfactant molecules, micelles or both? (2) How does unfolding of proteins in surfactant compare with "proper" unfolding in chemical denaturants? Recent work has highlighted the role of shared micelles, rather than monomers, below the critical micelle concentration (cmc) in promoting both protein denaturation and formation of higher order structures. Kinetic studies have extended the experimentally accessible range of surfactant concentrations to far above the cmc, revealing numerous different modes of denaturation by ionic surfactants below and above the cmc which reflect micellar properties as much as protein unfolding pathways. Uncharged surfactants follow a completely different denaturation strategy involving synergy between monomers and micelles. The high affinity of charged surfactants for proteins means that unfolding pathways are generally different in surfactants versus chemical denaturants, although there are common traits. Other issues are as follows: (3) Are there non-denaturing roles for SDS? (4) How reversible is unfolding in SDS? (5) How do solvent conditions affect the way in which surfactants denature proteins? The last three issues compare SDS with "proper" membranes. (6) Do anionic surfactants such as SDS mimic biological membranes? (7) How do mixed micelles interact with globular proteins? (8) How can mixed micelles be used to measure the stability of membrane proteins? The growing efforts to understand the unique features of membrane proteins have encouraged the development of mixed micelles to study the equilibria and kinetics of this class of proteins, and traits which unite globular and membrane proteins have also emerged. These issues emphasise the amazing power of surfactants to both extend the protein conformational landscape and at the same time provide convenient and reversible short-cuts between the native and denatured state for otherwise obdurate membrane proteins.     

Photosynthesis: the processing of redox signals in chloroplasts

Recent work identifies two kinases required for phosphorylation of proteins of chloroplast thylakoid membranes. One kinase, STN7, is required for phosphorylation of light-harvesting complex II; another, STN8, is required for phosphorylation of photosystem II. How do these kinases interact, what do they do, and what are they for?     

A rotary pumping model for helicase function of MCM proteins at a distance from replication forks

We propose an integrated model for eukaryotic DNA replication to explain the following problems: (1) How is DNA spooled through fixed sites of replication? (2) What and where are the helicases that unwind replicating DNA? (3) Why are the best candidates for replicative helicases, namely mini-chromosome maintenance (MCM) proteins, not concentrated at the replication fork? (4) How do MCM proteins spread away from loading sites at origins of replication? We draw on recent discoveries to argue that the MCM hexameric ring is a rotary motor that pumps DNA along its helical axis by simple rotation, such that the movement resembles that of a threaded bolt through a nut, and we propose that MCM proteins act at a distance from the replication fork to unwind DNA. This model would place DNA replication in a growing list of processes, such as recombination and virus packaging, that are mediated by ring-shaped ATPases pumping DNA by helical rotation.     

Stress responses: JAZ players deliver fusion and rhythm

How do stress-induced jasmonates cause genetic re-programming of plant cells? New research shows that the JAZ proteins provide the missing link.     

Functional competition within a membrane: Lipid recognition vs. transmembrane helix oligomerization

Binding of specific lipids to large, polytopic membrane proteins is well described, and it is clear that such lipids are crucial for protein stability and activity. In contrast, binding of defined lipid species to individual transmembrane helices and regulation of transmembrane helix monomer–oligomer equilibria by binding of distinct lipids is a concept, which has emerged only lately. Lipids bind to single-span membrane proteins, both in the juxta-membrane region as well as in the hydrophobic membrane core. While some interactions counteract transmembrane helix oligomerization, in other cases lipid binding appears to enhance oligomerization. As reversible oligomerization is involved in activation of many membrane proteins, binding of defined lipids to single-span transmembrane proteins might be a mechanism to regulate and/or fine-tune the protein activity. But how could lipid binding trigger the activity of a protein? How can binding of a single lipid molecule to a transmembrane helix affect the structure of a transmembrane helix oligomer, and consequently its signaling state? These questions are discussed in the present article based on recent results obtained with simple, single-span transmembrane proteins. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Self-organization and regulation of intrinsically disordered proteins with folded N-termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism.     

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the G_q subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the G_q subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.     

Cell polarity: PIN it down!

How do plants create and maintain cell polarity? Recent studies reveal a plant-specific mechanism, which links the static cellulose-based extracellular matrix to the dynamic localization of PIN auxin carrier proteins.     

Telomeres: not all breaks are equal

ATM, Rad50 and Mre11 have been shown to prevent telomere fusion in Drosophila, thereby extending the protective role of DNA damage checkpoint proteins to non-canonical telomeres formed without telomerase. How do these proteins help chromosomal termini escape fusion through 'repair' while promoting repair of induced DNA breaks?     

The molecular architecture of protein-protein binding sites

The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions?     

Germination of Spores of Bacillus Species: What We Know and Do Not Know

Spores of Bacillus species can remain in their dormant and resistant states for years, but exposure to agents such as specific nutrients can cause spores'' return to life within minutes in the process of germination. This process requires a number of spore-specific proteins, most of which are in or associated with the inner spore membrane (IM). These proteins include the (i) germinant receptors (GRs) that respond to nutrient germinants, (ii) GerD protein, which is essential for GR-dependent germination, (iii) SpoVA proteins that form a channel in spores'' IM through which the spore core''s huge depot of dipicolinic acid is released during germination, and (iv) cortex-lytic enzymes (CLEs) that degrade the large peptidoglycan cortex layer, allowing the spore core to take up much water and swell, thus completing spore germination. While much has been learned about nutrient germination, major questions remain unanswered, including the following. (i) How do nutrient germinants penetrate through spores'' outer layers to access GRs in the IM? (ii) What happens during the highly variable and often long lag period between the exposure of spores to nutrient germinants and the commitment of spores to germinate? (iii) What do GRs and GerD do, and how do these proteins interact? (iv) What is the structure of the SpoVA channel in spores'' IM, and how is this channel gated? (v) What is the precise state of the spore IM, which has a number of novel properties even though its lipid composition is very similar to that of growing cells? (vi) How is CLE activity regulated such that these enzymes act only when germination has been initiated? (vii) And finally, how does the germination of spores of clostridia compare with that of spores of bacilli?