The plasma membranes of living cells undergo constant recycling through the dynamic processes of membrane retrieval and membrane insertion. Through these endocytic and exocytic events, which include intermediate membrane transport pathways, the relative abundance of specific plasma membrane components and the secretion of molecules can be regulated. Complex mechanisms involving protein-protein interactions have evolved to coordinate such membrane trafficking processes to ensure that transported membranes are appropriately targeted to their specific destinations (36
). These mechanisms include directed movements on cytoskeletal tracks (39
) and the concerted actions of tethering proteins that anchor transport vesicles to cognate target membranes (27
). The membrane fusion step in exocytosis is highly dependent on proteins that join membranes in close proximity such that two separate lipid bilayers merge into one (16
). One class of proteins that function in this fusion step are the SNAREs (35
), of which the synaptic vesicle proteins synaptobrevin/VAMP (15
) and syntaxin 1 (1
), as well as SNAP-25 (13
) on the plasma membrane, are best characterized. Although SNAREs are clearly important for fusion, they may not be involved in directly executing the fusion reaction. The hypothesis that several other cofactors are necessary to complete fusion is supported by the observation that at least under some specialized conditions the deficiencies of certain SNAREs do not prevent fusion (5
). Furthermore, specialized exocytic systems such as those in neuronal synapses contain unique proteins that facilitate targeting or regulation of membrane fusion (28
). Thus, the molecular details of membrane fusion processes remain an active area of investigation.
Among a number of newly discovered proteins that have been implicated in the membrane targeting and fusion processes are the SM family of proteins (38
), the septins (12
), and RIM (40
) and associated proteins. A recent report has also suggested the involvement of a class V myosin, myo52, in fission yeast in mediating vacuole fusion under osmotic stress, providing the first link between an actin-based motor and homotypic membrane fusion (23
). This suggestion is particularly interesting in light of findings in our laboratory that a myosin I family member (Myo1c) is required for optimal insulin-stimulated translocation of intracellular membranes containing GLUT4 glucose transporters to the plasma membrane (3
). In this membrane trafficking system, GLUT4 recycles between intracellular and plasma membrane compartments and insulin acutely stimulates GLUT4 exocytosis through a phosphatidylinositol (PI) 3-kinase-dependent pathway (6
). The detailed mechanism by which GLUT4-containing membranes fuse with the plasma membrane requires interaction between syntaxin 4 (t-SNARE) (41
) and VAMP-2 (v-SNARE) (7
). However, it is not known which components or processes that function in the GLUT4 recycling pathway are directly downstream of PI 3-kinase signaling or require the myosin Myo1c.
The aim of the present studies was to characterize the role of Myo1c in the trafficking pathway of GLUT4-containing membranes and its relationship to PI 3-kinase-sensitive steps. Previous work had shown that the expression of high levels of Myo1c in cultured adipocytes enhances the extent to which GLUT4 is translocated to the plasma membrane in response to insulin (3
). In other recent studies, PI 3-kinase signaling was implicated in the fusion step of exocytosis of GLUT4-containing membranes (25
). Consistent with this hypothesis, we report here that the blockade of PI 3-kinase inhibits the fusion of GLUT4-containing membrane vesicles with the plasma membrane and causes the accumulation of these vesicles just beneath the cell surface. Remarkably, high expression of Myo1c could override the block in membrane fusion caused by PI 3-kinase inhibition when insulin is also present. These data suggest that Myo1c drives a process that promotes the fusion of GLUT4-containing vesicles with the plasma membrane.