Single-molecule reconstitution of mRNA transport by a class V myosin.

Molecular motors are instrumental in mRNA localization, which provides spatial and temporal control of protein expression and function. To obtain mechanistic insight into how a class V myosin transports mRNA, we performed single-molecule in vitro assays on messenger ribonucleoprotein (mRNP) complexes reconstituted from purified proteins and a localizing mRNA found in budding yeast. mRNA is required to form a stable, processive transport complex on actin--an elegant mechanism to ensure that only cargo-bound motors are motile. Increasing the number of localizing elements ('zip codes') on the mRNA, or configuring the track to resemble actin cables, enhanced run length and event frequency. In multi-zip-code mRNPs, motor separation distance varied during a run, thus showing the dynamic nature of the transport complex. Building the complexity of single-molecule in vitro assays is necessary to understand how these complexes function within cells.

Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece.

Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain.

Expression of Y53A-actin in Dictyostelium disrupts the cytoskeleton and inhibits intracellular and intercellular chemotactic signaling.

We showed previously that phosphorylation of Tyr(53), or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S., Hong, M. S., Levine, R. L., and Korn, E. D. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13694-13699; Liu, X., Shu, S., Hong, M. S., Yu, B., and Korn, E. D. (2010) J. Biol. Chem. 285, 9729-9739). We now show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, although individual amoebae chemotax normally. We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMP-induced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization. Trafficking of vesicles containing adenylyl cyclase A to the rear of the cell and secretion of the ACA vesicles are also inhibited. The actin cytoskeleton of cells expressing Y53A-actin is characterized by numerous short filaments, and bundled and aggregated filaments similar to the structures formed by copolymerization of purified Y53A-actin and wild-type actin in vitro. This disorganized actin cytoskeleton may be responsible for the inhibition of intracellular and intercellular cAMP signaling in cells expressing F-Y53A-actin.

Evidence for copper-dioxygen reactivity during alpha-synuclein fibril formation.

Alpha-synuclein (alpha-syn), a presynaptic protein implicated in Parkinson's disease, binds copper(II) ion (1:1) with submicromolar affinity in vitro. Insights on the molecular details of soluble- and fibrillar-Cu-alpha-syn are gained through X-ray absorption spectroscopy. Our results indicate that the copper coordination environment (3-to-4 N/O ligands, average Cu-ligand distance approximately 1.96 A) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-beta-sheet. Interestingly, we find that some population of Cu(II)-alpha-syn reduces to Cu(I)-alpha-syn in the absence of O(2). This autoreduction event appears diminished in the presence of O(2) suggestive of preceding Cu(I)/O(2) chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine cross-links in fibrillar samples.

Mutation of actin Tyr-53 alters the conformations of the DNase I-binding loop and the nucleotide-binding cleft.

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

Myo4p is a monomeric myosin with motility uniquely adapted to transport mRNA.

The yeast Saccharomyces cerevisiae uses two class V myosins to transport cellular material into the bud: Myo2p moves secretory vesicles and organelles, whereas Myo4p transports mRNA. To understand how Myo2p and Myo4p are adapted to transport physically distinct cargos, we characterize Myo2p and Myo4p in yeast extracts, purify active Myo2p and Myo4p from yeast lysates, and analyze their motility. We find several striking differences between Myo2p and Myo4p. First, Myo2p forms a dimer, whereas Myo4p is a monomer. Second, Myo4p generates higher actin filament velocity at lower motor density. Third, single molecules of Myo2p are weakly processive, whereas individual Myo4p motors are nonprocessive. Finally, Myo4p self-assembles into multi-motor complexes capable of processive motility. We show that the unique motility of Myo4p is not due to its motor domain and that the motor domain of Myo2p can transport ASH1 mRNA in vivo. Our results suggest that the oligomeric state of Myo4p is important for its motility and ability to transport mRNA.

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1.

Glycosylation of beta1 integrin (beta1) in the Golgi complex has been related to its function in multiple cell processes, e.g., invasiveness, matrix adhesion, and migration. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG) 1 and BIG2 activate human ADP-ribosylation factors (ARF) 1 and ARF3 by catalyzing the replacement of ARF-bound GDP with GTP to regulate Golgi vesicular transport. We show here a requirement for BIG1 (but not BIG2) in glycosylation and function of beta1. In HepG2 cells treated for 48 or 72 h with BIG1, but not BIG2, siRNA, both the amount and electrophoretic mobility of the initially 130-kDa beta1 were increased. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa beta1 was not seen. Peptide N-glycosidase F, but not endoglycosidase H, digestion converted all beta1 to an approximately 85-kDa (core protein) form. By electron microscopy, Golgi membranes in BIG1-depleted cells were less sharply defined than those in mock or BIG2 siRNA-treated cells, with more vesicle-like structures at the transface. Amounts of active RhoA-GTP also were decreased in such cells and restored by overexpression of HA-BIG1. Aberrant beta1 was present on the cell surface, but its function in cell spreading, adhesion, and migration was impaired. By immunofluorescence microscopy, BIG1 siRNA-treated cells showed less spreading and concentration of beta1 at the cell surface. These results indicate a previously unrecognized role for BIG1 in the glycosylation of beta1 by Golgi enzymes, which is critical for its function in developmental and other vital cell processes.

Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments.

Dictyostelium actin was shown to become phosphorylated on Tyr-53 late in the developmental cycle and when cells in the amoeboid stage are subjected to stress but the phosphorylated actin had not been purified and characterized. We have separated phosphorylated and unphosphorylated actin and shown that Tyr-53 phosphorylation substantially reduces actin's ability to inactivate DNase I, increases actin's critical concentration, and greatly reduces its rate of polymerization. Tyr-53 phosphorylation substantially, if not completely, inhibits nucleation and elongation from the pointed end of actin filaments and reduces the rate of elongation from the barbed end. Negatively stained electron microscopic images of polymerized Tyr-53-phosphorylated actin show a variable mixture of small oligomers and filaments, which are converted to more typical, long filaments upon addition of myosin subfragment 1. Tyr-53-phosphorylated and unphosphorylated actin copolymerize in vitro, and phosphorylated and unphosphorylated actin colocalize in amoebae. Tyr-53 phosphorylation does not affect the ability of filamentous actin to activate myosin ATPase.