Bariloche protein symposium argentine society for biochemistry and molecular biology

53
BIOCELL, 27 (Suppl. I), 2003
BC-C4.
NUCLEOSIDE DIPHOSPHATASE AND
GLYCOSYLTRANSFERASE ACTIVITIES CAN LOCALIZE
TO DIFFERENT SUBCELLULAR COMPARTMENTS
Cecilia D´Alessio and Armando J. Parodi.
Fundación Instituto Leloir, Buenos Aires, Argentina. E-mail:
[e-mail]
Nucleoside diphosphates generated by glycosyltransferases in cell
secretory pathways are converted into monophosphates to provide
substrates for antiport transport systems by which entrance of
nucleotide sugars from the cytosol is coupled to exit of nucleoside
monophosphates. The yeast Schizosaccharomyces pombe genome
encodes two enzymes with potential nucleoside diphosphatase
activity: Spgda1p and Spynd1p. Characterization of the
overexpressed enzymes showed that Spgda1p is a GDPase/UDPase
whereas Spynd1p is an apyrase. Individual disruption of their
encoding genes did not affect cell viability but disruption of both
genes was synthetically lethal. Disruption of Spgda1
+
 did not affect
Golgi N- or O-glycosylation whereas disruption of Spynd1
+
 affected
Golgi N-mannosylation. Subcellular fractionation showed that both
activities localize to the Golgi. Although no nucleoside
diphosphatase activity was detected in the ER, no ER accumulation
of misfolded glycoproteins occurred. N-glucosylation mediated by
the UDP-Glc:glycoprotein glucosyltransferase (GT) showed a
partial (35-50%) decrease in Spgda1 but was not affected in Spynd1
mutants. Results show that contrary to what has been assumed for
eukaryotic cells, in S. pombe nucleoside diphosphatase and
glycosyltransferase activities can localize to different subcellular
compartments. To test the hypothesis that ER-Golgi vesicle
transport might be involved in nucleoside diphosphate hydrolysis,
S. pombe conditional mutants in ER-Golgi transport bearing also
the  Spgda1 or Spynd1 genotype were constructed. The effect of
double mutations on GT-dependent ER glucosylation is currently
being assayed.
BC-C5.
DYNAMICS OF SNARE ASSEMBLY AND DISASSEMBLY
DURING HUMAN SPERM EXOCITOSIS
De Blas, G; Tomes, C; Yunes, R and Mayorga, L.
Laboratorio de Biología Celular y Molecular. Instituto de
Histología y Embriología (IHEM-CONICET), Facultad de
Ciencias Médicas, CC56, Universidad Nacional de Cuyo, 5500.
Mendoza-Argentina.  E-mail: [e-mail]
The acrosome reaction (AR) of spermatozoa is a complex, calcium-
dependent, regulated exocytosis. Fusion at multiple sites between
the outer acrosomal membrane and the cell membrane causes the
release of the acrosomal contents and the loss of the membranes
surrounding the acrosome. Acrosomal exocytosis is controlled by
a protein machinery that is conserved in organisms ranging from
yeast to human. SNARE proteins are essential components of this
fusion machinery. SNARE proteins exist in different
conformations. When packed in tight ternary complexes SNARE
proteins are resistant to cleavage by neurotoxins. In contrast,
monomeric or loosely assembled SNAREs are sensitive to
proteolysis. Here, we report a direct role for the SNARE complex
assembly and disassembly in the AR.  First, we show that the AR
is botulinum (BoNT) and tetanus (TeTx) toxin-resistant when the
system is unstimulated. Second, Rab3A promotes disassembly of
SNAREs that then become toxin sensitive. Third, SNAREs re-
assembly into BoNT/E and BoNT/B sensitive and TeTx-resistant
complexes before the efflux of acrosomal calcium.  We conclude
that SNAREs are not accessible to toxins before sperm activation,
suggesting a cis conformation under resting conditions. After
stimulation -but before acrosomal calcium release- SNAREs are
re-assembled in a loose trans configuration.
BC-C6.
THE CATION-DEPENDENT MANNOSE-6-PHOSPHATE
RECEPTOR IS NECESSARY  FOR DEVELOPMENT OF
RAT LIVER LYSOSOMES
Romano P, López C, Carvelli L, Sartor T and Sosa MA.
IHEM - CONICET- UNCuyo, Mendoza, Argentina. E-mail:
[e-mail]
Mannose-6-phosphate receptors (MPRs) play a role in selective
transport of acid hydrolases to lysosomes. Two types of MPR have
been described to date; the cation-dependent (CD-MPR) and the
cation-independent (CI-MPR) receptor, according to their
requirements for bivalent ions. In rodents, MPR expression varies
during perinatal development. In a previous study we demons-
trated a significant decrease of CI-MPR active binding sites (B
max
)
in rat liver at 10
th
 and 20
th
 day of birth. In contrast, the B
max
 for
CD-MPR increased significantly from the 10
th
 day and manteined
up to adulthood. From these findings we wondered if CD-MPR
can compensate the low activity of CI-MPR at these ages. We
measured some acid hydrolases at each age and their interaction
with membranes and observed that N-acetyl-â-D-glucosaminidase
(NAG), â -glucuronidase (GLU), and â -galactosidase (GAL)
activities (per mg of tissue) are higher from the day 10 after birth,
whereas the activity of á-mannosidase (MAN) remains unchanged.
By immunoblot we confirmed the tendency for MAN and NAG,
using specific antibodies. Moreover, the activities of NAG, GAL
and GLU bound to membranes correlated with those activities.
We also demonstrated that 60% of NAG bearing mannose-6-
phosphate was bound to CD-MPR in the livers at 10
th
 day, whilst
only 30% was bound to this receptor in adults. We concluded that
variations in lysosomal enzyme expression is mostly related to
CD-MPR during perinatal development, and this receptor may
substitute the CI-MPR at certain ages.
BC-C7.
ASSOCIATION OF TETRASPANIN CD63 WITH
INTEGRINS IN HUMAN DENDRITIC CELLS:
IMPLICATIONS FOR CELL MIGRATION. A
COMPARATIVE STUDY WITH OTHER TETRASPANINS
Mantegazza, Adriana R.; Barrio, Marcela and Mordoh, José.
Fundación Instituto Leloir, Capital Federal, Argentina. E-mail:
[e-mail]
Tetraspanins comprise a large group of ubiquitously expressed
membrane proteins implicated in many cellular functions. They
have the ability to associate with each other and with other surface
molecules, including integrins, MHC class I and II molecules and
intracellular signaling enzymes. Previously, we have demonstrated
that tetraspanins CD9, CD63, CD81, CD82 and CD151 are
expressed at the surface of human dendritic cells (DC), the most
potent antigen presenting cells. We have also shown that CD63
internalizes and follows the endocytic-MIICs route after
extracellular stimuli. We have further characterized the complexes
formed by CD63 on DC surface by FACS analysis and
immunoprecipitation techniques. We have found that after CD63
internalization upon binding of FC-5.01 monoclonal antibody
(Mab), the expression of tetraspanins CD9, CD81, CD82 and
CD151 as well as integrins CD29, CD11b, CD18 and 
α
5
,
diminished compared to control DC. Moreover, performing
chemotaxis assays on immature DC, we have observed that the
preincubation with Mabs directed against tetraspanins CD9, CD63,
CD81 and CD82 enhanced cell migration induced by MIP-1
α and
MIP-5 chemoattractants. We propose that the disruption of the
complexes between tetraspanins and integrins could be responsible
for the observed enhancement on DC migration. Besides, we have
observed a differential integrin expression between immature and
mature DC, which are known to differ in their migratory ability.

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