148 research outputs found
The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65
Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B. In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place. We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle. We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells. This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light. RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels. Following a 50–60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity. Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction. Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision
Myosin5a tail associates directly with Rab3A-containing compartments in neurons
Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of American Society for Biochemistry and Molecular Biology. The definitive version was published in Journal of Biological Chemistry, 286 (2011): 14352-14361, doi:10.1074/jbc.M110.187286.Myosin-Va (Myo5a) is a motor protein
associated with synaptic vesicles (SVs) but the
mechanism by which it interacts has not yet
been identified. A potential class of binding
partners are Rab GTPases and Rab3A is known
to associate with SVs and is involved in SV
trafficking. We performed experiments to
determine whether Rab3A interacts with
Myo5a and whether it is required for transport
of neuronal vesicles. In vitro motility assays
performed with axoplasm from the squid giant
axon showed a requirement for a Rab GTPase
in Myo5a-dependent vesicle transport.
Furthermore, mouse recombinant Myo5a tail
revealed that it associated with Rab3A in rat
brain synaptosomal preparations in vitro and
the association was confirmed by
immunofluorescence imaging of primary
neurons isolated from the frontal cortex of
mouse brains. Synaptosomal Rab3A was
retained on recombinant GST-tagged Myo5a
tail affinity columns in a GTP-dependent
manner. Finally, the direct interaction of
Myo5a and Rab3A was determined by
sedimentation v e l o c i t y analytical
ultracentrifugation using recombinant mouse
Myo5a tail and human Rab3A. When both
proteins were incubated in the presence of 1
mM GTPγS, Myo5a tail and Rab3A formed a
complex and a direct interaction was observed.
Further analysis revealed that GTP-bound
Rab3A interacts with both the monomeric and
dimeric species of the Myo5a tail. However, the
interaction between Myo5a tail and nucleotidefree
Rab3A did not occur. Thus, our results
show that Myo5a and Rab3A are direct binding
partners and interact on SVs and that the
Myo5a/Rab3A complex is involved in transport
of neuronal vesicles
The intracellular dynamic of protein palmitoylation
S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation
Creating Safety in Primary Care Practice with Electronic Medical Records Requires the Consideration of System Dynamics
Visualization of Melanosome Dynamics within Wild-Type and Dilute Melanocytes Suggests a Paradigm for Myosin V Function In Vivo
ER transport on actin filaments in squid giant axon: implications for signal transduction at synapse
Size, shape and mass of the oxygen-evolving photosystem II complex from the thermophilic cyanobacterium Synechococcus
The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth
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