Myosin V Transports Secretory Vesicles via a Rab GTPase Cascade and Interaction with the Exocyst Complex

Life Sciences Institute, Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2216, USA.
Developmental Cell (Impact Factor: 9.71). 12/2011; 21(6):1156-70. DOI: 10.1016/j.devcel.2011.10.009
Source: PubMed


Vesicle transport requires four steps: vesicle formation, movement, tethering, and fusion. In yeast, two Rab GTPases, Ypt31/32, are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4 and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2 and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation and remain attached until fusion.

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    • "In the charge reversal myo2-3DR- REKE strain, GFP-Sec4 polarization is significantly restored, though not to quite to the same degree as seen in wild-type cells. Exocyst component Sec15-GFP was also polarized in the myo2-3DR-REKE allele (Fig. S1 C), suggesting that defects in polarized transport related to this site may not be completely caused by a compromised Sec15p–Myo2p interaction as has been previously reported (Jin et al., 2011), but instead caused by motor misregulation. "
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    ABSTRACT: Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor. © 2015 Donovan and Bretscher.
    Full-text · Article · May 2015 · The Journal of Cell Biology
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    • "mice, demonstrating a functional relationship between these proteins (Fig. 6A,B). Both Rab8a and Rab11a are known to bind myosin Vb (Lapierre et al., 2001; Roland et al., 2007; Jin et al., 2011). Therefore, to determine whether myosin Vb protein is involved in the localisation of Rab11a, we compared small intestine samples from a microvillus atrophy patient and a healthy individual (Fig. 6C), as microvillus atrophy patients are known to have mutations in the myosin Vb gene (Müller et al., 2008). "
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    ABSTRACT: The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · Dec 2014 · Biology Open
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    • "Alternatively, axonal Exoc3 and TC10 mRNAs might subserve different functions in the regulation of membrane exocytosis. For example, in yeast the myosin V homologue, Myo2, interacts with the exocyst including Sec6p/Exoc346. The presence of Exoc3 mRNA in regenerating axons might therefore be a reflection of increased transport of secretory vesicles while local TC10 translation in developing axons triggers PPV tethering to specific secretion sites within the growth cones. "
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    ABSTRACT: The surface of developing axons expands in a process mediated by the exocyst complex. The spatio-temporal regulation of the exocyst is only partially understood. Here we report that stimulated membrane enlargement in dorsal root ganglion (DRG) axons is triggered by intra-axonal synthesis of TC10, a small GTPase required for exocyst function. Induced membrane expansion and axon outgrowth are inhibited after axon-specific knockdown of TC10 mRNA. To determine the relationship of intra-axonal TC10 synthesis with the previously described stimulus-induced translation of the cytoskeletal regulator Par3, we investigate the signalling pathways controlling their local translation in response to NGF. Phosphoinositide 3-kinase (PI3K)-dependent activation of the Rheb-mTOR pathway triggers the simultaneous local synthesis of TC10 and Par3. These results reveal the importance of local translation in the control of membrane dynamics and demonstrate that localized, mTOR-dependent protein synthesis triggers the simultaneous activation of parallel pathways.
    Full-text · Article · Mar 2014 · Nature Communications
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