Bariloche protein symposium argentine society for biochemistry and molecular biology

52
BIOCELL, 27 (Suppl. I), 2003
BE-C14.
CHARACTERIZATION OF THE SUBSTRATE BINDING
DOMAIN IN BACTERIAL ADP-GLUCOSE
PYROPHOSPHORYLASE
Erben, Esteban; Figueroa, Carlos; Fusari, Corina; Demonte, Ana;
Aleanzi, Mabel; Iglesias, Alberto A.
Grupo Enzimología Molecular, BBM, Fac. Bioq. Cs. Biol.,
UNLitoral. Paraje El Pozo, Santa Fe, 3000. Argentina. E-mail :
[e-mail]
Ballicora, Miguel; Preiss, Jack. Dept. Biochemistry & Mol. Biol.,
Michigan State University, East Lanzing, MI, USA.
ADPGlcPPase  is the key regulatory enzyme for the synthesis of
glycogen and starch in bacteria and plants, respectively. The
enzyme is allosterically regulated, with specificity for the regulator
depending on the source. Accumulated evidence strongly suggests
that ADPGlcPPases have a common folding pattern despite
different quaternary structure and specificity for activator. Domains
for substrate binding have been proposed in the predicted secondary
structure of the enzyme. To analyze the validity of the prediction
we characterized different mutants of bacterial ADPGlcPPase
randomly generated by the linker-scanning mutagenesis. Results
reinforce the structure model and agree with domains proposed
for binding of ATP and Glc1P. Data also suggest the occurrence of
conformational changes in the enzyme upon binding of the
activator. The latter was further analyzed by characterization of
site-directed mutants. It is suggested the occurrence of distinctive
changes induced by ADPGlcPPase specific activators.
Supported, in part ,by grants form ANPCyT (PICT’99 1-6074)
and Fundación Antorchas.
BC-C1.
DYNAMICS PROPERTIES OF THE GTPase RAB1 IN
LIVING CELLS
Pablo Monetta and Cecilia Alvarez.
Departamento de Bioquímica Clínica. Facultad de Ciencias
Químicas. Universidad Nacional de Córdoba. E-mail:
[e-mail]
Protein transport from the endoplasmic reticulum (ER) to the Golgi
requires the action of the GTPase Rab1.  Rab1 cycles between a
GDP and GTP-bound form and its activity is mediated by the
interaction of Rab1-GTP with different proteins.  However the
relationship between Rab1 dynamics and protein-protein
interaction is unclear. To study the dynamics of Rab1 we have
used time lapse microscopy of living cells (HeLa) transfected with
GFP-Rab1. The GFP-Rab1 localized predominantly to the Golgi
and to peripheral punctate structures. The majority of the GFP-
Rab1 labeled punctate structures underwent movements within a
limited area (less than 2 
µm in diameter) during time periods of 5
minutes. Only occasionally longer range, randomly directed
movements (aprox. 10 
µm) were observed.  The binding and release
kinetic of Rab1b to and from the Golgi membranes was analyzed
by fluorescence recovery after photobleaching (FRAP) in GFP-
Rab1b expressing cells. GFP-Rab1b bleached in the Golgi shows
rapidly recovery with a half time (t
1/2
) of 80 seconds. Taken together
our data suggest that: -Rab1 recruited in to peripheral structures
does not move towards the Golgi. -Rapid FRAP occurs as a results
of the exchange of Rab1 between Golgi membranes and the cytosol
and -Rab1 interacts simultaneously with diferent proteins localized
at the donor and acceptor compartments to control protein
trafficking at the ER-to-Golgi level.
BC-C2.
GANGLIOSIDE GLYCOSYLTRANSFERASES ORGANIZE
IN DISTINCT MULTIENZYME COMPLEXES IN CHO-K1
CELLS
Giraudo, Claudio G. and Maccioni, Hugo J.F.
CIQUIBIC (UNC-CONICET), Departamento de Química
Biológica, Facultad de Ciencias Químicas, UNC, Ciudad
Universitaria, 5000 Córdoba, Argentina. E-mail:
[e-mail]
Ganglioside synthesis is compartmentalized in the Golgi complex.
In most cells, glycosylation of LacCer, GM3 and GD3 to form
higher order species (GA2, GM2, GD2, GM1, GD1b) occurs in
distal aspects of the Golgi and the trans Golgi network, where the
involved transferases (GalNAcT and GalT2) form physical and
functional associations, while glycosylation of the simple species
LacCer, GM3 and GD3 occurs in more proximal Golgi
compartments. Herein we investigate if the involved transferases
(GalT1, SialT1 and SialT2) share the property of forming physical
associations. Co-immunoprecipitation experiments from CHO-K1
cell membranes expressing epitope tagged versions of these
enzymes indicate that they associate physically in a SialT1-
dependent manner, and that their N-terminal domains participate
in these interactions. Fluorescence microscopy in living cells
confirmed the interactions, showed Golgi localization of the
complexes and mapped their formation to the ER. No interactions
between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2
were detected. These results, and triple color imaging of Golgi
microvesicles reemerging the ER in Nocodazole treated cells
suggest that ganglioside synthesis is organized in distinct units
each formed by associations of particular glycosyltransferases, that
concentrate in different sub-Golgi compartments.
BC-C3.
AUTOPHAGY AS A NEW TARGET FOR CONTROL OF
COXIELLA AND MYCOBACTERIUM REPLICATION
Gutierrez, M.
1,3
; Vazquez, C.
1
; Munafó, D.
1
; Berón, W.
1
; Master,
S.
2
; Deretic, V.
2,3
 and Colombo, M.I.
1
1
IHEM- CONICET, Facultad de Ciencias Médicas, UNCuyo,
Mendoza, Argentina, and Depts. of 
2
Molecular Genetics and
Microbiology and 
3
Cell Biology and Physiology, University of
New Mexico HSC, Albuquerque, New México, USA.
[e-mail]
Many bacteria have evolved mechanisms to invade host cells and
survive in the intracellular environment. Coxiella burnetii
replicates in a compartment with lysosomal characteristics. We
have previously observed that C. burnetii localizes in a
compartment labeled by LC3, a protein that specifically localizes
in autophagic vacuoles. Interestingly, our results indicate that
autophagy induced an increase in the number of cells infected by
Coxiella. Furthermore, in cells overexpressing GFP-LC3 a marked
increase in the Coxiella-infection index was observed suggesting
that overexpression of proteins involved in the autophagic pathway
favors the generation of the Coxiella replicative niche.
Mycobacterium tuberculosis is known to interfere with the normal
maturation of the phagosome, preventing acidification. We have
observed that under autophagy conditions Mycobacterium-
containing phagosomes becomes more acidic and mature,
hampering the bacterial survival.
Our results suggest that autophagy can play opposing (beneficial
or detrimental to the pathogen) roles depending upon the organism.
Whereas Coxiella transits through autophagosomes as a strategy
for survival, induction of autophagy results in a less permissive
environment for Mycobacterium  replication. These observations
highlight the significance of the autophagic pathway as a new target
for intervention in infectious diseases.

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