Tubulin is subject to a special cycle of detyrosination͞tyrosination in which the C-terminal tyrosine of ␣-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persists in TTL null tissues, being present mainly in dividing TTL null cells where it originates from tubulin synthesis, but it is lacking in postmitotic TTL null cells such as neurons, which is apparently deleterious because early death in TTL null mice is, at least in part, accounted for by a disorganization of neuronal networks, including a disruption of the cortico-thalamic loop. Correlatively, cultured TTL null neurons display morphogenetic anomalies including an accelerated and erratic time course of neurite outgrowth and a premature axonal differentiation. These anomalies may involve a mislocalization of CLIP170, which we find lacking in neurite extensions and growth cones of TTL null neurons. Our results demonstrate a vital role of TTL for neuronal organization and suggest a requirement of Tyr-tubulin for proper control of neurite extensions.CLIP170 ͉ tubulin code
Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.
Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.
Abstract. The sequence of tubulin-tyrosine ligase (TTL), the enzyme catalyzing the ATP-dependent posttranslational addition of a tyrosine to the carboxyterminal end of detyrosinated ot-tubulin, has been determined. TTL from bovine and porcine brain was purified by immunoaffinity chromatography and extensively characterized by protein sequencing. Oligonucleotides derived from the protein sequence were synthesized and partial eDNA sequences were obtained using reversed transcribed brain mRNA in polymerase chain reactions. Polymerase chain reaction fragments were used to isolate a full-length eDNA clone from a randomly primed hgtl0 eDNA library obtained from embryonic porcine brain mRNA. Porcine TTL is encoded by 1,137 nucleotides corresponding to 379 amino acid residues. It has a molecular weight of 43,425 and a calculated isoelectric point of 6.51. Northern blot analysis revealed a surprisingly long mRNA (~ 6 kb in embryonic porcine brain). The protein sequence of TTL shares no extended homology with the sequences in the data banks. TTL contains a potential serine phosphorylation site for cAMP-dependent protein kinase (RKAS at positions 73 to 76). Residues 244 to 258 lie at the surface of the molecule. A rabbit antibody raised against a synthetic peptide corresponding to this sequence binds to native TTL. The same sequence contains the cleavage site for endoproteinase Glu-C (residue 248) previously shown to convert TTL into a nicked derivative in which the two fragments still form a tight complex but don't display enzymatic activity.
Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament‐associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface‐bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non‐motile (Domann et al., 1992, EMBO J., 11, 1981‐1990; Kocks et al., 1992, Cell, 68, 521‐531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C‐terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha‐actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline‐rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.
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