Association of L-glutamic acid decarboxylase to the 70-kDa heat shock protein as a potential anchoring mechanism to synaptic vesicles.
Recently we have reported that the membrane-associated form of the gamma-aminobutyric acid-synthesizing enzyme, l-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366-24371). In this report, several lines of evidence are presented to indicate that l-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD(65) as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD(65)-specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD(65) and GAD(67) are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in gamma-aminobutyric acid neurotransmission.
Hsu
C C
CC
Departments of Molecular Biosciences and Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Davis
K M
KM
Jin
H
H
Foos
T
T
Floor
E
E
Chen
W
W
Tyburski
J B
JB
Yang
C Y
CY
Schloss
J V
JV
Wu
J Y
JY
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D005968:Glutamate Decarboxylase / Q000276:immunology / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008565:Membrane Proteins; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; C088460:cysteine string protein; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase