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 that were reconstituted from purified proteins and a localizing mRNA found in budding yeast. mRNA is required to obtain 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 (“zipcodes”) on the mRNA, or configuring the track to resemble actin cables, enhanced run length and event frequency. In multi-zipcode mRNPs, motor separation distance varied during a run, 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

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.actin polymerization ͉ Dictyostelium ͉ phosphorylated actin T ransfer of Dictyostelium amoebae from nutrient to nonnutrient medium initiates a 24-hour developmental cycle (1) in which the amoebae aggregate and differentiate to form multicellular organisms that mature to fruiting bodies containing stable spores from which, when they are placed in nutrient medium, amoebae germinate. Several laboratories (2-4) reported tyrosine phosphorylation of actin [phosphotyrosine actin (pY-actin)] correlated with rearrangements of the actin cytoskeleton during the developmental cycle. pY-actin appears late in maturing spores, i.e., Ϸ24 h into the developmental cycle, reaches a maximum level at Ϸ36 h, at which time Ϸ50% of the actin is phosphorylated (3), remains constant for Ϸ20 days, at 22°C, and then decreases, disappearing entirely by 30 days, at which time the spores are no longer viable (3). When viable spores are placed in nutrient medium, pY-actin is dephosphorylated, with a half-life of Ϸ5 min (3), before spore swelling and germination (2, 4).Although vegetative amoebae in nutrient medium contain little or no pY-actin (5, 6), phosphorylation transiently increases (for Ϸ20-25 min) when amoebae are transferred from nonnutrient to nutrient medium (7), with concurrent changes in cell shape, for example, loss of pseudopods, rounding up of the previously elongated cells, and weakened adherence to the substratum (7). Tyrosine phosphorylation also occurs when vegetative amoebae in nutrient medium are exposed to phenylarsine oxide (PAO) (5), an inhibitor of phosphotyrosine phosphatase, or are subjected to stress, for example, inhibition of oxidative phosphorylation (8, 9) or elevated temperature (9), with parallel changes in cell shape similar to the changes that occur when cells are transferred from nonnutrient to nutrient medium.Importantly, phosphorylation occurs uniquely at Tyr-53 (9). Thus, phosphoryl...

α-Synuclein (α-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-α-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 ~1.96 Å) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-β-sheet. Interestingly, we find that some population of CuII-α-syn reduces to CuI-α-syn in the absence of O2. This autoreduction event appears diminished in the presence of O2 suggestive of preceding CuI/O2 chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine crosslinks in fibrillar samples.

Glycosylation of ␤1 integrin (␤1) 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 ␤1. 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 ␤1 were increased. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa ␤1 was not seen. Peptide Nglycosidase F, but not endoglycosidase H, digestion converted all ␤1 to an Ϸ85-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 vesiclelike structures at the transface. Amounts of active RhoA-GTP also were decreased in such cells and restored by overexpression of HA-BIG1. Aberrant ␤1 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 ␤1 at the cell surface. These results indicate a previously unrecognized role for BIG1 in the glycosylation of ␤1 by Golgi enzymes, which is critical for its function in developmental and other vital cell processes.adhesion ͉ ADP-ribosylation factor ͉ migration I ntegrins are heterodimeric transmembrane glycoproteins comprising diverse ␣-and ␤-subunits. Most of the integrins involved in focal adhesion (FA) formation are members of the ␤ 1 or ␤ 3 families. The ␤1 integrin (␤1) subunit pairs with at least 12 different ␣-subunits to form transmembrane adhesion receptors for extracellular matrix (ECM) proteins, e.g., collagen, fibronectin, and laminin (1). Integrin binding of ECM initiates intracellular signaling, including RhoA-dependent events, such as development of lamellipodia, actin stress fibers, and FAs that act in cell spreading and adhesion (2).The function of ␤1 depends on its accurate glycosylation catalyzed by enzymes located in morphologically and biochemically distinct cisternae of the Golgi system. Mature ␤1 is transported to the cell surface, where it mechanically links plasma membrane adhesion complexes to the actin cytoskeleton for bidirectional transmembrane as well as intracellular signaling. Numerous processes, e.g., embryogenesis, wound healing, and tumorigenesis, are regulated in this way (3, 4); factors that can affect ␤1 glycosylation and transport include Ki-Ras (5), transforming growth factor-␤ (6), and HEMCAM/gicerin (7). Treatment of HeLa cells with ceramide or brefeldin A caused fragmentation of the Golgi complex and inhibition of ␤1 glycosylation (8). Accumulated data indicate the importance of ␤1 N-glycosylation in cell a...

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

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.A s summarized in the accompanying paper (1), Acanthamoeba myosin II (AMII) is a typical class II myosin with two heavy chains and two pairs of light chains (2, 3). The coiled-coil helical tails of the heavy chains terminate with a 27-residue nonhelical tailpiece, 1483PSSRGGSTRGASARGASVRAGSARAEE1509, which has a pattern of four contiguous repeats of XXSXR (4). Previous work had shown that phosphorylation of two or more of these serines (residues 1489, 1494, 1499, and 1504) correlates with, and was assumed to be responsible for, inactivation of AMII's actin-activated MgATPase activity (5, 6). Also, it had been concluded that only filamentous AMII has actin-activated MgATPase activity (7, 8), and it was inferred that the ATPase activity is regulated by a change in the conformation of the bipolar minifilaments (9-11). However, detailed studies by the Pollard laboratory found no significant differences in either the polymerization properties or electron microscopic images of minifilaments of phosphorylated and dephosphorylated myosins (12-16).The earlier studies were carried out with purified endogenous myosin. To avoid possible complications in enzymatic and structural studies caused by the partial phosphorylation of purified endogenous myosin and incomplete dephosphorylation by phosphatase, we initiated studies on the enzymatic activity and structure of expressed recombinant wild-type, truncated, and mutant myosins before and after phos...

from dmantADP bound to myosin V 1IQ (MV 1IQ) or Dicty myosin II S1 (Dicty S1) in the presence of 1 mM MgCl 2 . We found that dmantADP bound to acto-MV 1IQ contained two lifetime components (8 and 3 ns). Timeresolved anisotropy studies of the two lifetime components in myosin V reveal that the long lifetime component was more immobilized (correlation time = 12 ns) while the short lifetime component was highly dynamic (correlation time = 0.4 ns). Interestingly, Dicty S1 contained a single lifetime component (8 ns). The two conformations of the myosin V active site may allow Mg 2þ to more efficiently bind and reduce key steps in the ATPase cycle such as ADP release. Overall, our results suggest that differences in the structural dynamics of the active site of myosins may play a role in their dependence on free Mg 2þ , which could explain why Mg 2þ differentially alters the motile and force generating properties of myosins.