Article #2 Summary
DepartmentCell and Systems Biology
CSB428H1F Article #2: Chuang et al. SARA and Rhodopsin Transport
OS discs are renewed whereby rhodopsin-laden vesicles in the OS axonemal cytoplasm fuse with
nascent discs abundant in PI3P and FYVE domain protein SARA interacts with PI3P, rhodopsin and
syntaxin 3 SNARE to regulate this process.
Rhodopsin’s C’ tail has an address signal to OS and also binds SARA.
FYVE domain is a conserved double zinc finger motif that binds PI3P specifically.
SARA (smad anchor for receptor activation) has FYVE and is located on EE.
SARA interacts with rhodopsin C’
In yeast two hybrid and GST pull down assay, rhodopsin C’ interacts with human SARA. Ab for
rhodopsin also co-immuniprecipitated endogenous mouse SARA.
In HEK cells, SARA colocalized with EEA1 and rhodopsin on EE.
SARA is enriched in axonemeal vesicles
In rods, SARA is found in punctate EE in apical cytoplasm of IS and basal portion of proximal
axoneme including the connecting cilium and basal body.
Rhodopsin was found throughout OS.
SARA-PI3P interaction is necessary for rhodopsin delivery to OS
A PI3P probe consisted of double FUVE finger of Hrs that binds PI3P specifically.
In rods, PI3P was found in OS especially proximal portion.
PI3K Vps34 was found only at proximal portion of OS towards base, likely on discs, but not in IS.
Rods transfected with human-rhodopsin with FYVESARA-RFP overexpressed show h-rhodopsin was
mislocalized to cell body and synapses and FYVESARA-RFP was localized to proximal region
showing FYVESARA-RFP out-competes endogenous SARA for PI3P binding.
Endogenous rhodopsin was reduced in OS and increased in IS. Here FYVESARA-RFP is DN.
Ectopic 3XFYVEEEA1-GFP had the same effect, showing PI3P-FYVE interaction is necessary.
Arrestin method: arrestin is localized to cell body/synapse in dark rods but upon light stimulus it
translocates to OS and binds activated rhodopsin. Arrestin also binds mislocalized rhodopsin.
FYVESARA-RFP lead to arrestin-GFP trapped in cell body/synapse in rods exposed to light.
FYVESARA-RFP transfected rods have disorganized OS with tubules/vesicles and multi-vesicular
bodies of various sizes at basal OS. OS axoneme and ciliary membrane was swollen. OS PM
ruptured as small vesicles accumulated in extracellular space between IS and OS. IS was normal.
SARA suppression impair rhodopsin targeting to OS
SARA-sh/RFP knocks down SARA, which caused mislocalization of h-rhodopsin and accumulation
of tubulo-vesicles and multi-vesicular bodies at basal OS and in swollen axoneme resembling
FYVESASA overexpression. Transfection with sh-SARA resistant construct rescued normal h-
rhodopsin delivery to OS.
Syntaxin 3 SNARE binds SARA
Syntaxin 3 was enriched in basal OS axoneme and resembles that of SARA indicating it is
distributed on vesicles. Syntaxin 3 was also found on PM and EE with SARA in HEK cells.
SARA binds syntaxin 3 in GST pull down assays. Other component of SNARE core also co-
precipitated with SARA in HEK cells.
Rods with syntaxin 3-sh/GFP knockdown caused h-rhodopsin mislocalization to cell body/synapse.