Targeting of membrane and secretory proteins to the apical domain in epithelial cells.

The sorting and delivery of membrane and secretory proteins to the apical domain of epithelial cells remain rich fields for investigation. The different classes of apical membrane proteins have distinct targeting signals within their structure, but most signals have not yet been identified. The single, transmembrane proteins have been studied in some detail and their routing to the apical surface differs among epithelial cells. This difference can be exploited in the search for signals. Additionally, the different secretory responses of cells to microtubule disruption may reveal further insights into the dynamics of the apical domain.     

Intracellular protein trafficking defects in human disease

Secretory proteins and integral membrane proteins travel through the secretory pathway to a variety of destinations. Their targets are often specified by signals in the amino acid sequence or signals added post-translationally. The KDEL sequence that retains soluble proteins in the endoplasmic reticulum and the mannose 6-phosphate group of lysosomal enzymes are well-characterized examples of targeting signals; other signals are less well understood. Given the complexity and importance of the intracellular trafficking pathways, it is perhaps not surprising that mutations that affect the trafficking of proteins are associated with some human genetic diseases.     

Phosphorylation-dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.     

Spatial organization of intracellular Ca2+ signals

The ability of Ca(2+), the simplest of all intracellular messengers, selectively to regulate so many cellular behaviours is due largely to the complex spatiotemporal organization of intracellular Ca(2+) signals. Most signalling pathways, including those that culminate in Ca(2+) signals, comprise sequences of protein-protein interactions linked by diffusible messengers. Using specific examples to illustrate key principles, we consider the roles of both components in defining the spatial organization of Ca(2+) signals. We discuss evidence that regulation of most Ca(2+) channels by Ca(2+) contributes to controlling the duration of Ca(2+) signals, to signal integration and, via Ca(2+)-induced Ca(2+) release, to defining the spatial spread of Ca(2+) signals. We distinguish two types of protein-protein interaction: scaffolds that allow rapid local transfer of diffusible messengers between signalling proteins, and interactions that directly transfer information between signalling proteins. Store-operated Ca(2+) entry provides a ubiquitous example of the latter, and it serves also to illustrate how Ca(2+) signals can be organized at different levels of spatial organization - from interactions between proteins to interactions between organelles.     

Vectorial delivery of macromolecules into cells using peptide-based vehicles

The ability to direct the import of therapeutic agents into cells and target them to specific organelles would greatly enhance their functional efficacy. The available spectrum of peptide-based import signals and intracellular routing signals might provide practical solutions towards achieving a guided or vectorial delivery of molecules. Multiple cell-targeting signals and routing domains can be efficiently displayed on branched peptides. These constructs are typically nonimmunogenic in the absence of adjuvant and can be easily assembled using solid phase synthesis. The vectorial delivery of larger complexes, however, will necessitate the development of alternate templates that favor the optimal presentation of all functional signals.     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

Intracellular copper routing: the role of copper chaperones

Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the 'copper chaperones'. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.     

Regulation of the nucleocytoplasmic trafficking of viral and cellular proteins by ubiquitin and small ubiquitin-related modifiers

Nucleocytoplasmic trafficking of many cellular proteins is regulated by nuclear import/export signals as well as post-translational modifications such as covalent conjugation of ubiquitin and small ubiquitin-related modifiers (SUMOs). Ubiquitination and SUMOylation are rapid and reversible ways to modulate the intracellular localisation and function of substrate proteins. These pathways have been co-opted by some viruses, which depend on the host cell machinery to transport their proteins in and out of the nucleus. In this review, we will summarise our current knowledge on the ubiquitin/SUMO-regulated nuclear/subnuclear trafficking of cellular proteins and describe examples of viral exploitation of these pathways.     

Simulated Evolution of Signal Transduction Networks

Signal transduction is the process of routing information inside cells when receiving stimuli from their environment that modulate the behavior and function. In such biological processes, the receptors, after receiving the corresponding signals, activate a number of biomolecules which eventually transduce the signal to the nucleus. The main objective of our work is to develop a theoretical approach which will help to better understand the behavior of signal transduction networks due to changes in kinetic parameters and network topology. By using an evolutionary algorithm, we designed a mathematical model which performs basic signaling tasks similar to the signaling process of living cells. We use a simple dynamical model of signaling networks of interacting proteins and their complexes. We study the evolution of signaling networks described by mass-action kinetics. The fitness of the networks is determined by the number of signals detected out of a series of signals with varying strength. The mutations include changes in the reaction rate and network topology. We found that stronger interactions and addition of new nodes lead to improved evolved responses. The strength of the signal does not play any role in determining the response type. This model will help to understand the dynamic behavior of the proteins involved in signaling pathways. It will also help to understand the robustness of the kinetics of the output response upon changes in the rate of reactions and the topology of the network.     

Runnin' with the Dvl: proteins that associate with Dsh/Dvl and their significance to Wnt signal transduction

Wnt proteins transmit myriad intercellular signals crucial for the development and homeostasis of metazoan animals from Hydra to human. Abnormal Wnt signaling causes a growing number of diseases, including cancer and osteoporosis. Depending on the context, a given Wnt signal may denote: cell proliferation or apoptosis; cell fate determination, differentiation, or stem cell maintenance; a variety of changes in cell behavior; and/or coordinated interactions with its neighbors. Which event(s) occur in Wnt-responsive cells depends critically on the ability of Dishevelled (Dsh)/Dvl proteins to interpret distinct types of intracellular, receptor-generated stimuli and transmit them to at least two distinct sets of effector molecules, all while apparently ignoring a third type of Wnt-generated Ca(2+) signal. The three conserved domains present in Dsh/Dvl proteins uniquely function in each Wnt pathway, in part by association with 18 (and counting) Dsh/Dvl-associated proteins. The latest data suggest that Dsh/Dvl proteins organize dynamic, pathway-specific subcellular signaling complexes that ensure correct information routing, signal amplification, and dynamic control through feedback regulation. The biochemical and cell biological mechanisms by which Dsh/Dvl proteins accomplish these remarkable tasks remain obscure.     

Rapid responses: Receptor-like kinases directly regulate the functions of membrane transport proteins in plants

Receptor-like kinases (RLKs) are a large group of plant-specific transmembrane proteins mainly acting as receptors or co-receptors of various extracellular signals. They usually turn extracellular signals into intracellular responses via altering gene expression profiles. However, recent studies confirmed that many RLKs can physically interact with diverse membrane-localized transport proteins and regulate their activities for speedy responses in limited tissues or cells. In this minireview, we highlight recent discoveries regarding how RLKs can work with membrane transport proteins collaboratively and thereby trigger cellular responses in a precise and rapid manner. It is anticipated that such regulation broadly presents in plants and more examples will be gradually revealed when in-depth analyses are conducted for the functions of RLKs.     

Striated muscle proteins are regulated both by mechanical deformation and by chemical post-translational modification

All cells sense force and build their cytoskeleton to optimize function. How is this achieved? Two major systems are involved. The first is that load deforms specific protein structures in a proportional and orientation-dependent manner. The second is post-translational modification of proteins as a consequence of signaling pathway activation. These two processes work together in a complex way so that local subcellular assembly as well as overall cell function are controlled. This review discusses many cell types but focuses on striated muscle. Detailed information is provided on how load deforms the structure of proteins in the focal adhesions and filaments, using α-actinin, vinculin, talin, focal adhesion kinase, LIM domain-containing proteins, filamin, myosin, titin, and telethonin as examples. Second messenger signals arising from external triggers are distributed throughout the cell causing post-translational or chemical modifications of protein structures, with the actin capping protein CapZ and troponin as examples. There are numerous unanswered questions of how mechanical and chemical signals are integrated by muscle proteins to regulate sarcomere structure and function yet to be studied. Therefore, more research is needed to see how external triggers are integrated with local tension generated within the cell. Nonetheless, maintenance of tension in the sarcomere is the essential and dominant mechanism, leading to the well-known phrase in exercise physiology: “use it or lose it.”     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Mechanical aspects of cell shape regulation and signaling

Physical forces play a critical role in cell integrity and development, but little is known how cells convert mechanical signals into biochemical responses. This mini-review examines potential molecular mediators like integrins, focal adhesion proteins, and the cytoskeleton in the context of a complex cell structure. These molecules-when activated by cell binding to the extracellular matrix-associate with the skeletal scaffold via the focal adhesion complex. Vinculin is presented as a mechanical coupling protein that contributes to the integrity of the cytoskeleton and cell shape control, and examples are given of how mechanical signals converge into biochemical responses through force-dependent changes in cell geometry and molecular mechanics.     

Hidden localization motifs: naturally occurring peroxisomal targeting signals in non-peroxisomal proteins

Background

Can sequence segments coding for subcellular targeting or for posttranslational modifications occur in proteins that are not substrates in either of these processes? Although considerable effort has been invested in achieving low false-positive prediction rates, even accurate sequence-analysis tools for the recognition of these motifs generate a small but noticeable number of protein hits that lack the appropriate biological context but cannot be rationalized as false positives.

Results

We show that the carboxyl termini of a set of definitely non-peroxisomal proteins with predicted peroxisomal targeting signals interact with the peroxisomal matrix protein receptor peroxin 5 (PEX5) in a yeast two-hybrid test. Moreover, we show that examples of these proteins - chicken lysozyme, human tyrosinase and the yeast mitochondrial ribosomal protein L2 (encoded by MRP7) - are imported into peroxisomes in vivo if their original sorting signals are disguised. We also show that even prokaryotic proteins can contain peroxisomal targeting sequences.

Conclusions

Thus, functional localization signals can evolve in unrelated protein sequences as a result of neutral mutations, and subcellular targeting is hierarchically organized, with signal accessibility playing a decisive role. The occurrence of silent functional motifs in unrelated proteins is important for the development of sequence-based function prediction tools and the interpretation of their results. Silent functional signals have the potential to acquire importance in future evolutionary scenarios and in pathological conditions.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.