N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks.

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis, and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and cofilin, recent work also implicates posttranslational acetylation or arginylation of the actin N terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated elongation, Arp2/3-mediated nucleation, and severing by cofilin. Altogether, this study shows that the nature of the N terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge.

Regulation of actin isoforms in cellular and developmental processes.

Actin is one of the most abundant and essential intracellular proteins that mediates nearly every form of cellular movement and underlies such key processes as embryogenesis, tissue integrity, cell division and contractility of all types of muscle and non-muscle cells. In mammals, actin is represented by six isoforms, which are encoded by different genes but produce proteins that are 95-99 % identical to each other. The six actin genes have vastly different functions in vivo, and the small amino acid differences between the proteins they encode are rigorously maintained through evolution, but the underlying differences behind this distinction, as well as the importance of specific amino acid sequences for each actin isoform, are not well understood. This review summarizes different levels of actin isoform-specific regulation in cellular and developmental processes, starting with the nuclear actin's role in transcription, and covering the gene-level, mRNA-level, and protein-level regulation, with a special focus on mammalian actins in non-muscle cells.

Dynamics of intracellular stress-induced tRNA trafficking.

Stress is known to induce retrograde tRNA translocation from the cytoplasm to the nucleus but translocation kinetics and tRNA-spatial distribution have not been characterized previously. We microinject fluorescently-labeled tRNA into living cells and use confocal microscopy to image tRNA spatial distribution in single cells at various levels of starvation and to determine translocation rate constants. Retrograde tRNA translocation occurs reversibly, within minutes after nutrition depletion of the extracellular medium. Such nutritional starvation leads to down-regulation of tRNA nuclear import and nearly complete curtailment of its nuclear export. Nuclear tRNA accumulation is suppressed in cells treated with the translation inhibitor puromycin, but is enhanced in cells treated with the microtubule inhibitor nocodazole. tRNA in the cytoplasm exhibits distinct spatial distribution inconsistent with diffusion, implying that such distribution is actively maintained. We propose that tRNA biological complexes and/or cytoplasmic electric fields are the likely regulators of cytoplasmic tRNA spatial distribution.

Reduced passive force in skeletal muscles lacking protein arginylation.

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.

Protein Arginylation: Milestones of Discovery.

Posttranslational modifications have emerged in recent years as the major biological regulators responsible for the orders of magnitude increase in complexity during gene expression and regulation. These "molecular switches" affect nearly every protein in vivo by modulating their structure, activity, molecular interactions, and homeostasis ultimately regulating their functions. While over 350 posttranslational modifications have been described, only a handful of them have been characterized. Until recently, protein arginylation has belonged to the list of obscure, poorly understood posttranslational modifications, before the recent explosion of studies has put arginylation on the map of intracellular metabolic pathways and biological functions. This chapter contains an overview of all the major milestones in the protein arginylation field, from its original discovery in 1963 to this day.

Preparation of ATE1 Enzyme from Native Mammalian Tissues.

Early studies of protein arginylation preceded the wide availability of recombinant protein expression and relied heavily on the fractionation of proteins from native tissues. This procedure has been developed in 1970 by R. Soffer, in the wake of arginylation discovery in 1963. This chapter follows the detailed procedure originally published by R. Soffer in the 1970, adapted from his article in consultation with R. Soffer, H. Kaji, and A. Kaji.

Assaying ATE1 Activity in Yeast by ß-Gal Degradation.

In the 1980s, it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta-galactosidase (beta-Gal) because its level can be easily measured using standardized colorimetric assays. Here, we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species.

Development of New Tools for the Studies of Protein Arginylation.

Studies of posttranslational modifications present many unique challenges, stemming from their role as the major drivers of biological complexity. Perhaps the most immediate challenge to researchers working on virtually any posttranslational modification is the shortage of reliable easy-to-use tools that can enable massive identification and characterization of posttranslationally modified proteins, as well as their functional modulation in vitro and in vivo. In the case of protein arginylation, which utilizes charged Arg-tRNA that is also used by the ribosomes, detection and labeling of arginylated proteins is especially difficult, because of the necessity of distinguishing these proteins from the products of conventional translation. As of now, this difficulty remains the major obstacle to new researchers entering the field. This chapter discusses some of the strategies for developing antibodies for arginylation detection, as well as some general considerations for development of other tools for studies of arginylation.

Assaying Intracellular Arginylation Activity Using a Fluorescent Reporter.

In this chapter, we present a simplified version of the method described in Chapter 9 of this book, adapted for fast and convenient evaluation of intracellular arginylation activity in live cells. As in the previous chapter, this method utilizes a GFP-tagged N-terminal ß-actin peptide transfected into cells as a reporter construct. Arginylation activity can then be evaluated by harvesting the reporter-expressing cells and analyzing them directly by Western blot using an arginylated ß-actin antibody and a GFP antibody as an internal reference. While absolute arginylation activity cannot be measured in this assay, different types of reporter-expressing cells can be directly compared, and the effect of genetic background or treatment can be evaluated. For its simplicity and broad biological application, we felt this method merited presentation here as a separate protocol.

Bacterial Expression and Purification of Recombinant Arginyltransferase (ATE1) and Arg-tRNA Synthetase (RRS) for Arginylation Assays.

Here, we describe the procedure for the expression and purification of recombinant ATE1 from E. coli. This method is easy and convenient and can result in one-step isolation of milligram amounts of soluble enzymatically active ATE1 at nearly 99% purity. We also describe a procedure for the expression and purification of E. coli Arg-tRNA synthetase essential for the arginylation assays described in the next two chapters.

Preparation of tRNAArg for Arginylation Assay by In Vitro Transcription.

This chapter describes the preparation of tRNAArg by in vitro transcription. tRNA produced by this method can be efficiently utilized for in vitro arginylation assays, following aminoacylation with Arg-tRNA synthetase, either directly during the arginylation reaction or separately to produce the purified preparation of Arg-tRNAArg. tRNA charging is described in other chapters of this book.

Enzymatic Aminoacylation of tRNAArg Using Recombinant Arg-tRNA Synthetase.

This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g., for measuring the kinetics or determining the effect of different compounds and chemicals on the reaction. In such cases, tRNAArg can be pre-charged with Arg and purified away from the RARS enzyme prior to arginylation.

Assaying ATE1 Activity In Vitro.

Here, we describe a standard arginyltransferase assay in vitro using bacterially expressed purified ATE1 in a system with a minimal number of components (Arg, tRNA, Arg-tRNA synthetase, and arginylation substrate). Assays of this type have first been developed in the 1980s using crude ATE1 preparations from cells and tissues and then perfected recently for the use with bacterially expressed recombinant protein. This assay represents a simple and efficient way to measure ATE1 activity.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

High-Throughput Arginylation Assay in Microplate Format.

Here, we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for the identification of small molecule inhibitors and activators of ATE1, high-volume analysis of AE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified 2 compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.

Analysis of Arginylated Peptides by Subtractive Edman Degradation.

During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable, but highly depends on the purity and abundance of samples and can become misleading unless a highly purified highly arginylated protein can be obtained. Here, we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples. This method can also apply to the analysis of other posttranslational modifications.

Identification of Arginylated Proteins by Mass Spectrometry.

Here, we describe the method for the identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides and then expanded to the side chain modification which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra. These methods can be used with both complex and purified protein samples and, to date, constitute the only reliable way to confirm arginylation at a particular site on a protein or peptide.

Preparation of an Enriched tRNAArg Fraction for Arginylation by Expression in E. coli.

The method described here provides a fast and efficient way to obtain an enriched preparation of tRNA of interest, which is also posttranscriptionally modified by the intracellular machinery of the host cells, E. coli. While this preparation also contains a mixture of total E. coli tRNA, the enriched tRNA of interest is obtained in high yields (milligram) and is highly efficient for biochemical assays in vitro. It is routinely used in our lab for arginylation.

Differential N-terminal processing of beta and gamma actin.

Cytoplasmic beta- and gamma-actin are ubiquitously expressed in every eukaryotic cell. They are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-termini, making it difficult to dissect their individual regulation. Here, we analyzed actin from cultured cells and tissues by mass spectrometry and found that beta, unlike gamma actin, undergoes sequential removal of N-terminal Asp residues, leading to truncated actin species found in both F- and G-actin preparations. This processing affects up to ∼3% of beta actin in different cell types. We used CRISPR/Cas-9 in cultured cells to delete two candidate enzymes capable of mediating this type of processing. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration. Our results demonstrate previously unknown isoform-specific actin regulation that can potentially affect actin functions in cells.

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles.

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).