Structure And Function Of Unconventional Myosins and CAR

非常规肌球蛋白和CAR的结构和功能

• 批准号: 7321512
• 负责人: JOHN A HAMMER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7321512
• 关键词: Structure; Function; Unconventional; Myosins; CAR

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.