| dc.description.abstract |
<p>Rod photoreceptors are specialized neurons responsible for capturing photons and
translating visual information into electrical signals. Visual signal transduction
in rods is confined to the unique outer segment organelle, a modified primary cilium
consisting of a stack of hundreds of flattened disc membranes enveloped by a single
plasma membrane. By concentrating important signaling molecules on disc membranes,
the outer segment provides an ideal biochemical environment for the production of
vision with high sensitivity and temporal resolution.</p><p>This dissertation focuses
primarily on a molecular dissection of two multifunctional outer segment proteins,
R9AP and rhodopsin, and also reassesses the localization of Glut1, a third protein
formerly believed to reside in the outer segment. All three experimental lines relied
on in vivo expression of novel protein constructs in vertebrate rods using several
gene delivery strategies: conventional transgenics, retinal electroporation, and retinal
infection with recombinant adeno-associated virus.</p><p>The tail-anchored protein
R9AP, in conjunction with RGS9-1 and G-beta5, comprises the transducin GTPase activating
complex, which catalyzes the rate-limiting step in rod photoresponse recovery. In
addition to maximizing the enzymatic activity of the complex, R9AP is responsible
for both the post-translational stability and outer segment targeting of RGS9-1-G-beta5.
We investigated the mechanism behind R9AP's poorly understood function in protecting
RGS9-1-G-beta5 from proteolysis and found that it is performed simply by recruiting
the complex to cellular membranes and can be entirely dissociated from R9AP's outer
segment targeting function. Furthermore, we demonstrated that replacement of R9AP's
transmembrane domain with a lipid anchor preserves the ability of the GTPase activating
complex to function in outer segments.</p><p>Rhodopsin, the visual pigment of rods,
has a second important, yet poorly defined, function as a rod outer segment building
block: outer segments disc membranes fail to form in the absence of rhodopsin. Our
goal was to identify the molecular features of rhodopsin mechanistically involved
in outer segment morphogenesis by designing artificial membrane proteins that could
fully substitute for rhodopsin in performing this function. We observed that rhodopsin's
C-terminal VXPX outer segment targeting motif is unnecessary for outer segment disc
formation since it could be replaced with a targeting motif from an unrelated protein,
peripherin. Furthermore, we obtained surprising evidence that rhodopsin's role in
this process is limited to providing an abundance of transmembrane protein material
to disc membranes.</p><p>Finally, while attempting to find a targeting motif to substitute
for the VXPX motif of rhodopsin, we made an unexpected observation that the facilitative
glucose transporter Glut1, long thought to reside in the outer segment, is actively
excluded from this organelle. This revises our understanding of the energy flow in
rods by showing that the outer segment is entirely dependent on the inner segment
for its energy supply.</p>
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