The enzymatic conjugation of arginine to the N-termini proteins is a part ubiquitin-dependent N-end rule pathway protein degradation. In mammals, three N-terminal residues—aspartate, glutamate, and cysteine—are substrates for arginylation. mouse ATE1 gene encodes family Arg-tRNA-protein transferases (R-transferases) that mediate We constructed ATE1-lacking strains found −/− embryos die with defects in heart development angiogenic remodeling early vascular plexus. Through biochemical...

Posttranslational arginylation is critical for mouse embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects the identity of proteins arginylated in vivo are unknown. We found that beta-actin was to regulate actin filament properties, localization, lamella formation motile cells. Arginylation apparently represents a step N-terminal processing needed functioning vivo. Thus, posttranslational single protein target can intracellular function, inducing global...

The mammalian cytoskeletal proteins β- and γ-actin are highly homologous, but only β-actin is amino-terminally arginylated in vivo, which regulates its function. We examined the metabolic fate of exogenously expressed nonarginylated actin isoforms. Arginylated γ-actin, unlike β-, was unstable selectively ubiquitinated degraded vivo. This instability regulated by differences nucleotide coding sequence between two isoforms, conferred different translation rates. translated more slowly than...

Posttranslational arginylation is critical for embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects the identity of proteins arginylated in vivo are largely unknown. Here we report a global analysis this modification on protein level identification 43 highly specific sites. Our data demonstrate that unlike previously believed, can occur any N-terminally exposed residue likely defined by structural recognition motif surface, it preferentially affects number...

The N-end rule relates the in vivo half-life of a protein to identity its N-terminal residue. underlying ubiquitin-dependent proteolytic system, called pathway, is organized hierarchically: aspartate and glutamate (and also cysteine metazoans) are secondary destabilizing residues, that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), arginine, primary We isolated cDNA encoding 516-residue mouse R-transferase, ATE1p, found two species, termed...

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how affects actin cytoskeleton, we investigated biochemical properties and structural organization of filaments wild-type arginyltransferase (Ate1) knockout cells. We found that Ate1 results a dramatic reduction polymer levels vivo accompanied by corresponding increase monomer level. Purified nonarginylated has altered polymerization properties, from cells...

Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action Met-aminopeptidases expose arginylated site. Here, we tested possibility linkage midchain sites within intact protein targets and found many proteins in vivo are modified on side chains Asp Glu unconventional chemistry carboxy rather than amino groups at target sites. Such...

The N-end rule relates the in vivo half-life of a protein to identity its N-terminal residue. asparagine and glutamine are tertiary destabilizing residues, that they enzymatically deamidated yield secondary residues aspartate glutamate, which conjugated arginine, primary arginine substrate is bound by Ubr1-encoded E3alpha, E3 component ubiquitin-proteasome-dependent pathway. We describe construction analysis mouse strains lacking asparagine-specific amidase (Nt(N)-amidase), encoded Ntan1...

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise organs tissues throughout body. Recent studies protein arginylation implicated this poorly understood posttranslational modification in functioning actin cytoskeleton culture. Knockout arginyltransferase (Ate1) mice leads embryonic lethality severe heart defects are reminiscent migration–dependent phenotypes seen...

Talin is a large scaffolding molecule that plays major role in integrin-dependent cell–matrix adhesion. A for talin cell–cell attachment through cadherin has never been demonstrated, however. Here, we identify novel calpain-dependent proteolytic cleavage of results the release 70-kD C-terminal fragment, which serves as substrate posttranslational arginylation. The intracellular levels this fragment closely correlated with formation adhesions, and localized to cadherin-containing contacts....

β‐ and γ‐cytoplasmic actin are nearly indistinguishable in their amino acid sequence, but encoded by different genes that play non‐redundant biological roles. The key determinants drive functional distinction unknown. Here, we tested the hypothesis β- γ-actin functions defined nucleotide, rather than using targeted editing of mouse genome. Although previous studies have shown disruption β-actin gene critically impacts cell migration embryogenesis, demonstrate here generation a lacking...

Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape poorly understood. The spectrin-based membrane skeleton maintains the biconcave of erythrocytes, but whether spectrins also determine nonerythroid cells is less clear. We show βIII spectrin hippocampal cortical from rodent embryos both sexes distributed throughout somatodendritic compartment particularly enriched neck base...

β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type nearly identical at the amino acid level but play vastly different roles vivo. Their essential embryogenesis mesenchymal migration critically depend on nucleotide sequences of their genes, rather than sequences; however, it is unclear which gene elements underlie this effect. Here we address specific role coding sequence actins' intracellular functions, using stable polyclonal populations immortalized mouse embryonic...

Arginylation is the posttranslational addition of arginine to a protein by arginyltransferase-1 (ATE1). Previous studies have found that ATE1 targets multiple cytoskeletal proteins, and Ate1 deletion causes defects, including reduced cell motility adhesion. Some these defects been linked actin arginylation, but role other arginylated proteins has not studied. Here, we characterize tubulin arginylation its in microtubule cytoskeleton. We identify ATE1-dependent ⍺-tubulin at E77. Ate1−/− cells...