Structure And Function Of Unconventional Myosins and CAR

肌凝蛋白Va轻链DYNLL2已被提出作为连接肌凝蛋白与特定货物的适配器。我们在肌凝蛋白Va重链中定位了DYNLL2的结合位点。共同纯化和下拉实验表明，重链含有一个DYNLL2结合位点，该位点位于肌凝蛋白？S中心线圈域。重要的是，外显子B，一个选择性剪接的三氨基酸外显子，是这个结合位点的一部分，我们在全长myosin Va的背景下表明，这个外显子是DYNLL2-myosin Va相互作用所必需的。我们研究了DYNLL2结合对肌凝蛋白Va重链片段结构的影响，该片段包含DYNLL2结合位点和侧翼序列，其中只有部分被强烈预测形成盘绕线圈。圆二色性测量揭示了dynll2诱导的二聚体肌球蛋白片段二级结构的变化，这与a-螺旋线圈含量的增加是一致的。此外，DYNLL2的结合极大地稳定了这种重链片段，防止热变性。分析性超离心得到DYNLL2与二聚体肌球蛋白片段相互作用的明显关联常数为~3 × 106 M-1。总之，这些数据表明，肌凝蛋白Va重链的选择性剪接控制着DYNLL2-肌凝蛋白Va的相互作用，DYNLL2的结合改变了肌凝蛋白Va的一部分结构。S线圈域。这些结果表明，外显子B可能对天然肌凝蛋白Va的构象和调节折叠以及与某些货物的相互作用有重要影响。

The myosin Va light chain DYNLL2 has been proposed to function as an adaptor to link the myosin to certain cargo. We mapped the binding site for DYNLL2 within the myosin Va heavy chain. Co-purification and pull-down experiments showed that the heavy chain contains a single DYNLL2 binding site and that this site resides within a discontinuity in the myosin?s central coiled-coil domain. Importantly, exon B, an alternatively spliced, three-amino-acid exon, is a part of this binding site, and we show in the context of full-length myosin Va that this exon is required for DYNLL2-myosin Va interaction. We investigated the effect of DYNLL2 binding on the structure of a myosin Va heavy chain fragment that contains the DYNLL2 binding site plus flanking sequence, only parts of which are strongly predicted to form coiled-coil. Circular dichroism measurements revealed a DYNLL2-induced change in the secondary structure of this dimeric myosin fragment that is consistent with a gain in a-helical coiled-coil content. Moreover, the binding of DYNLL2 considerably stabilizes this heavy chain fragment against thermal denaturation. Analytical ultracentrifugation yielded an apparent association constant of ~3 x 106 M-1 for the interaction of DYNLL2 with the dimeric myosin fragment. Together, these data show that alternative splicing of the myosin Va heavy chain controls DYNLL2-myosin Va interaction and that DYNLL2 binding alters the structure of a portion of the myosin?s coiled-coil domain. These results suggest that exon B could have a significant impact on the conformation and regulatory folding of native myosin Va, as well as on its interaction with certain cargos. The contractile vacuole (CV) complex is a specialized intracellular membrane compartment that serves as the osmoregulatory organelle in protozoa. In Dictyostelium, this compartment is composed of an interconnected network of tubules and cisternae or bladders. These membranes accumulate excess water (e.g. rain water) that has entered the cell by osmosis by pumping protons and most likely bicarbonate into their lumen. The resulting ion gradient draws the excess water out of the cytoplasm and into the lumen. The swollen bladders that are generated expel this excess water from the cell through transient fusion pores in the plasma membrane. Elegant studies using quick-freeze, deep etch electron microscopy, interference reflection microscopy, and fluorescent dyes (e.g. FM-64) or proteins (e.g. GFP-tagged dajumin) to visualize CV membrane dynamics in vivo have revealed a number of important aspects about the CV system in Dictyostelium. First, the tubules and bladders that comprise the system are highly dynamic and very pleiomorphic. Second, tubules and bladders are rapidly interconvertible. Third, CV membranes do not mix with the endosomal/lysosomal membrane system or with the plasma membrane during the process of water expulsion. What has emerged from these studies is a working definition of a CV membrane cycle in Dictyostelium in which swollen, mature bladders contact the fusion pore in the plasma membrane, water is discharged from the cell, the collapsed bladder membrane folds up into a tight knot immediately under the plasma membrane, this knot of membrane rapidly transforms into tubules that radiate out across the actin rich cortex, and these tubules fuse with each other and with immature bladders during the filling phase to create new mature bladders. These cortical event are seen best in time lapse confocal images of the ventral surface of adherent cells, as this configuration places a large area of the plasma membrane and subjacent actin-rich cortex within a single focal plane. The close association of CV membranes with the actin-rich cortex, and the dramatic motility of CV tubules along the cortex have lead to the suggestion that CV membranes recruit some type of myosin. We show that the Dictyostelium type V myosin myoJ is targeted to CV membranes and is responsible for their steady state association with the actin-rich cortex. Moreover, we show that myo J drives the tubulation of collapsed bladder membranes along the cortex following water discharge. Finally, the steady state accumulation of CV membranes around the MTOC seen in myoJ null cells forced us to visualize CV membrane dynamics in the middle of the cell as well as along its ventral surface. These images revealed that the tubules emanating from collapsed bladders move not only on actin in the plane of the membrane but bidirectionally along microtubules between the cortex and the microtubule organizing center (MTOC) adjacent to the nucleus. From this we conclude that myoJ cooperates with plus and minus end-directed microtubule motors to drive the proper distribution and function of the CV complex in Dictyostelium. Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. We now show that this interaction serves to sequester CP in an inactive state. More strikingly, CARMIL uncaps actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not antagonize the capping activity of gelsolin nor of CapG. While full length (FL) CARMIL (residues 1-1121) possesses these activities, C-terminal fragments like GST-P (940-1121) that contain CARMIL?s CP binding site are at least ten times more active. We localized the full activities to the C-terminal 51 residues (51aa; 1071-1121), which contains a stretch of 25 residues that are highly conserved among protozoan, fly, worm, and vertebrate CARMIL proteins (CARMIL Homology domain-3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-capping activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP ~20 fold more tightly than does FL-CARMIL. This observation, together with the elevated anti-capping protein activities of C-terminal fragments relative to FL-CARMIL, suggests that FL-CARMIL might exist primarily in an auto-inhibited state. Consistent with this, proteolytic cleavage of FL-CARMIL with thrombin generated an ~14-kDa C-terminal fragment that expresses full sequestering and uncapping activities. We propose that, following some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly. Capping protein is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing method for purifying native Capping Protein from tissues and recombinant Capping Protein from bacteria is a time labor-intense process that involves a number of traditional chromatographic steps to achieve a homogeneous preparation of the protein. We devised a one-step purification of Acanthamoeba Capping Protein from amoeba extracts and recombinant mouse Capping Protein from E. Coli extracts using as an affinity matrix GST fusion proteins containing the Capping Protein binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for Capping Protein purification should facilitate the in vitro analysis of Capping Protein structure, function and regulation.