Abstract: | Membrane recycling and remodeling contribute to multiple cellular
functions, including cell fusion events during myogenesis. We have identified
a tripartite motif (TRIM72) family member protein named MG53 and defined its
role in mediating the dynamic process of membrane fusion and exocytosis in
striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif
at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell
imaging of green fluorescent protein-MG53 fusion construct in cultured
myoblasts showed that although MG53 contains no transmembrane segment it is
tightly associated with intracellular vesicles and sarcolemmal membrane. RNA
interference-mediated knockdown of MG53 expression impeded myoblast
differentiation, whereas overexpression of MG53 enhanced vesicle trafficking
to and budding from sarcolemmal membrane. Co-expression studies indicated that
MG53 activity is regulated by a functional interaction with caveolin-3. Our
data reveal a new function for TRIM family proteins in regulating membrane
trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in
membrane organization occur as myoblast fusion allows the formation of
multinucleated muscle fibers. In addition to cell fusion events,
differentiation of myotubes involves establishment of specialized membrane
structures (1,
2). The transverse tubular
invagination of sarcolemmal membrane and the intracellular membrane network
known as the sarcoplasmic reticulum are two highly organized membrane
architectures in cardiac and skeletal muscle. Establishment of these intricate
membrane compartments requires extensive remodeling of the immature myoblast
membranes. Dynamic membrane remodeling also contributes to many physiologic
processes in mature muscle, including Ca2+ signaling, trafficking
of glucose transporter (GLUT4), and other membrane internalization events
involving caveolae structures
(3-6).
Although defects in membrane integrity have been linked to various forms of
muscular dystrophy (7,
8), the molecular machinery
regulating these specific membrane recycling and remodeling events in striated
muscle is not well defined.The large tripartite motif
(TRIM)5 family of
proteins is involved in numerous cellular functions in a wide variety of cell
types. Members of this protein family contain signature motifs that include a
RING finger, a zinc binding moiety (B-box), and a
coiled coil structure (RBCC), which invariably comprise
the amino-terminal domain of TRIM family members
(9). The carboxyl-terminal
sequence of TRIM proteins is variable; in some cases a subfamily of TRIM
proteins contains a SPRY domain, a sequence first observed in the ryanodine
receptor Ca2+ channel in the sarcoplasmic reticulum membrane of
excitable cells (10).
Extensive studies have revealed that protein-protein interactions in the
cytosol mediate the defined functions of TRIM proteins. For example, the
ubiquitin E3 ligase enzymatic activity of several TRIM family members requires
the B-box motif (11,
12). Recent studies have also
indicated a role for TRIM proteins in defense against events involving
membrane penetration, such as protection against infection by various viruses,
including human immunodeficiency virus
(13-15).
Although most of the studies concentrate on the cytosolic action of TRIM,
limited reports have investigated the role of TRIM proteins in membrane
signaling or recycling.We have previously established an immunoproteomics approach that allows
definition of novel components involved in myogenesis, Ca2+
signaling, and maintenance of membrane integrity in striated muscle
(16). Using this approach, we
have shown that junctophilin is a structural protein that establishes
functional communication between sarcoplasmic reticulum and transverse tubule
membranes at triad and dyad junctions in striated muscle
(17-19).
Further studies identified mitsugumin 29, a synaptophysin-related protein that
is essential for biogenesis of triad membrane structures and Ca2+
signaling in skeletal muscle
(20,
21). Screening of this
immunoproteomics library led to the recent identification of MG53, a
muscle-specific TRIM family protein
(22). Domain homology analysis
revealed that MG53 contains the prototypical RBCC motifs plus a SPRY domain at
the carboxyl terminus. Genetic knock-out and functional studies reveal that
MG53 nucleates the assembly of the sarcolemmal membrane repair machinery to
restore cellular integrity following acute damage to the muscle fiber
(22).Here we present evidence illustrating that MG53, in contrast to other known
TRIM proteins, can localize to intracellular vesicles and the sarcolemmal
membrane. A functional interaction between MG53 and caveolin-3, another
muscle-specific protein, plays an essential role in regulating the dynamic
process of membrane budding and exocytosis in skeletal muscle. |