SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
GAG attachment alters APP intracellular transport, potentially influencing its processing and Aβ
generation.
1987
Methyl esterification of retinal proteins is essential for rod-mediated vision.
J. R. Christiansen 1 , S. Kolandaivelu 1 , M. O. Bergo 2 , S. G. Young 3 , V. Ramamurthy 1,4 ;
1 Ophthalmology, West Virginia University, Morgantown, WV, 2 Cancer Center Sahlgrenska,
University of Gothenburg, 3 eDepartments of Medicine and Human Genetics, David Geffen
School of Medicine University of California, Los Angeles, 4 Biochemistry, West Virginia University
Background: Proteins ending in a “CAAX” box are first prenylated at their c-terminal cysteine
and then RAS-converting enzyme 1 (RCE1) cleaves the final three amino acids before
isoprenylcysteine methyltransferase (ICMT) catalyzes the methyl esterification of the newly
prenylated cysteine residue. Prenylation is crucial for a protein’s ability to interact with
membrane domains. The contribution of the final two steps to a protein’s localization and
function are variable and depends on the protein being studied. We recently demonstrated the
importance of RCE1-mediated proteolysis in phototransduction. In the absence of Rce1,
photoreceptors do not function and rapidly degenerate. To determine if the effects seen in the
retina conditional knockout of Rce1 were related to the lack of proteolysis, lack of carboxyl
methylation, or both we analyzed the retina of mice with reduced levels of Icmt.
Methods: We utilized the hypomorphic Icmt fl allele as a tool to generate animals expressing
various levels of Icmt in the retina. Electroretinogram (ERG) recordings were used to analyze
visual function of littermate animals with a range of Icmt levels. Morphology and
phototransduction protein expression profiles were investigated by immunofluorescence and
immunoblotting respectively.
Results: ERG recordings displayed delayed photoreceptor cell responses and reduced
downstream electrical responses to light stimuli. The delayed photoreceptor cell response
correlates to changes in Icmt message levels in the retina. In agreement with delayed
photoreceptor cell responses, critical phototransduction protein levels were also altered. To
investigate the cause of the reduced downstream neuronal responses, we are currently verifying
the development and proper synaptic stratification of inner retinal neurons.
Conclusion: Efficient coupling of signal transduction in photoreceptor neurons requires methyl
esterification of a phototransduction protein. In comparison with Rce1 CKO, we did not observe
rapid retinal degeneration or loss of visual function. At low light levels, the visual response was
reduced corresponding to reductions in Icmt. In agreement with the loss of visual response
phototransduction protein turnover was also increased.
1988
Turnover of amyloid precursor protein carboxy terminal fragment beta (C99) in lysosomal
compartments.
A. Rivera-Dictter 1 , H. Bustamante 1 , V. Muñoz 1 , V. Cavieres 1 , G. A. Mardones 1 , J. S. Bonifacino 2 ,
P. V. Burgos 1 ; 1 Universidad Austral de Chile Sch Med, Valdivia, Chile, 2 CBMP, National Institute
of Child Health and Human Development, NIH, Bethesda, MD
Proteolytic processing of the amyloid precursor protein by β-secretase generates C99, which
subsequently is cleaved by γ-secretase, yielding the amyloid β peptide (Aβ). C99 contains
within its cytosolic tail key signal motifs for its delivery to lysosomal compartments. Our aim was
to investigate lysosomal turnover of C99 in a mechanism independent of γ-secretase cleavage.
H4 neuroglioma cells stably expressing C99-EGFP-WT were analyzed in pulse-chase
experiments with cycloheximide, in the absence or presence of γ-secretase inhibitors testing
several conditions in order to: 1) block delivery to lysosomes and 2) disrupt lysosomal function.