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MECHANISMS OF NEUROTRANSMITTER STORAGE AND RELEASE 
Reinhard Jahn, Ph.D., Associate Investigator 
Dr. Jahn and his colleagues are interested in the 
mechanisms by which neurons store and release neu- 
rotransmitters. Neurotransmitters are stored in syn- 
aptic vesicles that are concentrated in nerve termi- 
nals and are released by Ca^^-dependent exocytosis 
upon stimulation. Exocytosis occurs at specialized 
zones of the presynaptic plasma membrane (active 
zones). It is an extremely rapid event, indicating 
that all components required for membrane fusion 
are preassembled at the site of release. The mecha- 
nisms of membrane recycling are less well under- 
stood, but it is likely that it occurs via coated vesi- 
cles and early endosomes as intermediate stages. 
During the past several years. Dr. Jahn's group has 
concentrated on a biochemical characterization of 
the membrane of synaptic vesicles. These studies 
provide the basis for a molecular characterization of 
individual steps of synaptic vesicle recycling, a line 
of research currently under development. The char- 
acterization of synaptic vesicle membrane compo- 
nents, carried out largely in collaboration with Dr. 
Thomas Siidhof (HHMI, University of Texas South- 
western Medical Center at Dallas) and Dr. Pietro De 
Camilli (HHMI, Yale University) led to the identifi- 
cation of several unique protein families that are 
specifically localized on neuronal synaptic vesicles 
and related microvesicles in neuroendocrine cells. 
In addition, uptake and storage of amino acid neuro- 
transmitters by synaptic vesicles was studied in 
some detail, focusing on the bioenergetic aspects of 
these processes. 
Recently the work has concentrated on two of the 
synaptic vesicle proteins, synaptotagmin and rab3A. 
Synaptotagmin, which was cloned and sequenced in 
collaboration with Dr. Siidhof, possesses two 
unique domains that are homologous to the regula- 
tory C2 domain of protein kinase C. This domain is 
responsible for Ca^^ and phospholipid binding. A 
newly generated monoclonal antibody was used to 
affinity purify synaptotagmin to homogeneity and 
test its ability to interact with Ca^* and phospho- 
lipids. The results show clearly that synaptotagmin 
is capable of high-affinity Ca^"^ binding, provided 
that acidic phospholipids are present. The interac- 
tion apparently involves the formation of a ternary 
complex between synaptotagmin, Ca^^, and phos- 
pholipid vesicles. This capacity is maintained when 
synaptotagmin itself is incorporated into liposomes. 
Under these conditions, Ca^^ at physiologically rele- 
vant concentrations induces vesicle aggregation but 
not fusion. Dr. Jahn and his colleagues assume that 
synaptotagmin is functioning as a Ca^^ receptor at 
the center of the fusion complex between synaptic 
vesicle membrane and plasma membrane. Mem- 
brane fusion itself presumedly involves additional 
proteins. The current efforts are concentrated on re- 
constituting exocytotic membrane fusion in vitro. 
The rab3A protein is a representative of a subfam- 
ily of small GTP-binding proteins related to ras. It is 
specifically localized to synaptic vesicles. The rab 
proteins have been shown to be highly specific for 
intermediates in intracellular membrane traffic, 
where they are involved in the orderly and consecu- 
tive execution of individual trafficking steps. Dur- 
ing exo-endocytosis, rab3A dissociates from the vesi- 
cle membrane and reassociates again at a later stage 
of the membrane cycle. Thus rab3A appears to be 
specific for vesicles ready to undergo exocytosis but 
lacking from endocytic vesicles. In agreement with 
this hypothesis, rab3A is absent from the membrane 
of coated vesicles. Recently the studies were ex- 
tended to include the small GTP-binding proteins 
rab5 and rab7, which have been shown by others to 
be specific for early and late endosomes, respec- 
tively. Both these proteins are present in nerve ter- 
minals, where they are located on synaptic vesicle- 
derived membranes. However, the rab5- and 
rab7-containing membrane pool only partially over- 
laps with that containing rab3A. Thus these small 
GTP-binding proteins may serve as convenient tools 
408 
