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

SUNDAY
after the membrane adaptor proteins dissociated from the patches. We were surprised to find
that actin patches also assembled slowly in these cofilin mutant cells. Adaptor proteins such as
End4p and Pan1p accumulated and persisted at the endocytic sites more than 10 times longer
than in wild type cells, followed by delayed put persistent recruitment of activators of Arp2/3
complex, including WASP and myosin I. We propose that severing by cofilin normally produces
short actin filaments that diffuse out of actin patches and stimulate Arp2/3 complex in adjacent
patches by serving as the mother filaments that initiate the autocatalytic branching reaction.
Additional feedback mechanisms seem to prolong the early steps in the pathway until the later
steps are executed.
1983
Structural and functional characterization of cargo-binding sites of the µ4-subunit of
adaptor protein complex 4.
B. H. Ross 1 , E. A. Corales 1 , Y. Lin 1 , J. H. Hurley 2 , J. S. Bonifacino 3 , P. V. Burgos 1 , G. A.
Mardones 1 ; 1 Universidad Austral de Chile Sch Med, Valdivia, Chile, 2 LMB, National Institute of
Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 3 CBMP, National Institute of
Child Health and Human Development, NIH, Bethesda, MD
Adaptor protein (AP) complexes assist protein trafficking by playing key roles in the selection of
cargo molecules to be sorted in post-Golgi compartments. The medium-sized subunit (µ1-µ4) of
the four heterotetrameric AP complexes recognizes YXXØ-sequences (Ø is a bulky hydrophobic
residue), which are sorting signals in transmembrane proteins. A conserved region, the µ2binding
site, mediates recognition of YXXØ-signals. Recently we found that a non-canonical
YXXØ-signal binds to a distinct µ4-binding site of the AP-4 complex. In this study we aimed to
determine the functionality of both binding sites on the recognition of this non-canonical YXXØsignal.
We used site-directed mutagenesis, yeast-two hybrid (Y2H) analyses, isothermal titration
calorimetry (ITC), and X-ray crystallography. Substitutions in either of both binding sites on µ4
abrogated binding to the APP-tail in Y2H experiments. Further characterization by ITC showed
no binding only with the R283D substitution at the µ4-binding site, in contrast with a decrease in
binding affinity with the substitution D190A at the µ2-binding site. We solved the crystal
structure of the C-terminal domain of the D190A mutant of the µ4 subunit bound to the noncanonical
YXXØ-signal. This structure showed no significant difference compared to that of
wild-type µ4 subunit. Our mutational, biochemical and structural analyses established the role of
the µ4-binding site for the non-canonical YXXØ-signal.
FONDECYT 1100896
1984
Regulation of the RalGAP Complex by Akt-Catalyzed Phosphorylation.
D. Leto 1,2 , X-W. Chen 1,2 , A. Burk 1,2 , T. Xiong 1,3 , G. Yu 1,2 , A. Saltiel 1,2 ; 1 University of Michigan,
Ann Arbor, MI, 2 Life Sciences Institute, University of Michigan, Ann Arbor, MI, 3 Department of
Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
In response to feeding, the anabolic hormone insulin increases glucose uptake into adipocytes
by stimulating exocytosis of vesicles containing the facilitative glucose transporter, Glut4. The
small vesicle-localized G protein, RalA, is rapidly and transiently activated by insulin in an Aktdependent
manner. When bound to GTP, RalA facilitates recognition of Glut4 vesicles at the
plasma membrane by interacting with the exocyst, an 8-subunit tethering complex. We have
previously shown that in the absence of insulin, RalA is retained in a largely inactive state by a
RalGAP Complex comprised of a regulatory subunit, RGC1, and a catalytic subunit, RGC2, that
contains a GAP domain with specific activity toward RalA. Activation of this GTPase by insulin
requires inhibition of the RalGAP complex. Here, we show that insulin stimulates Akt-catalyzed