SECRETORY PATHWAYS IN NEURONS 
Thomas C. Sudhof, M.D., Associate Investigator 
Information is transferred between neurons at 
the synapse. Here neurotransmitters are released 
by the presynaptic cell and upon binding to re- 
ceptors evoke a variety of postsynaptic responses. 
The presynaptic nerve terminal is a highly com- 
plex cellular structure. It synthesizes neurotrans- 
mitters and packages them into synaptic vesicles. 
These are targeted to the release site, secrete the 
neurotransmitters by exocytosis in a tightly regu- 
lated manner, and recycle. An understanding of the 
biological mechanisms by which neurotransmission 
occurs is dependent on understanding what hap- 
pens during neurotransmitter release. The study of 
the release of neurotransmitters operates at the in- 
terface between cell biology and neurobiology; its 
results will be important for concepts about pro- 
tein sorting and organelle biogenesis in eukaryotic 
cells and for elucidating synaptogenesis and the 
regulation of neurotransmission in the nervous 
system. 
Dr. Sudhof and his colleagues have embarked on 
a program to study the synaptic vesicle as the cen- 
tral organelle in presynaptic function. The first aim 
is to define the proteins of synaptic vesicles, their 
structures and localization. This has been achieved 
to the extent that several of these proteins have 
been purified, cloned, and expressed. The next aim 
is to determine how these proteins participate in 
the function of synaptic vesicles. Initial studies have 
provided insight into how synaptic vesicles may be 
formed and how their proteins may interact at the 
synaptic vesicle surface. 
The predominant type of synaptic vesicle in ver- 
tebrate brain is the small translucent synaptic vesi- 
cle. These vesicles accumulate at the presynaptic 
density and primarily contain amino acid neuro- 
transmitters, but not neuropeptides. The mem- 
branes of the small translucent synaptic vesicles ex- 
hibit a characteristic pattern of proteins. There are 
four major extrinsic membrane proteins that are 
phosphorylated upon neuronal stimulation. These 
extrinsic phosphoproteins, which are closely re- 
lated to each other, are synapsins la, lb, Ila, and lib. 
Previous work has established that synapsins la and 
lb bind to several elements of the cytoskeleton and 
that the four synapsins together account for —10% 
of the total synaptic vesicle membrane protein. In 
collaboration with Dr. Paul Greengard, Dr. Sudhof 
and his colleagues characterized the synapsins by 
molecular cloning and expression. The synapsins 
were found to be the differentially spliced products 
of two genes, one of which encodes synapsins la 
and lb; the other encodes synapsins Ila and lib. 
The amino-terminal regions of all four synapsins 
are highly homologous to each other and share a 
similar phosphorylation site for cAMP-dependent 
protein kinase and Ca^^ calmodulin-dependent 
protein kinase I. The carboxyl-terminal regions di- 
verge in a manner such that each synapsin has a dif- 
ferent set of shared or individual domains. The four 
synapsins were distributed differentially among syn- 
apses in rat brain. Their structures, distributions, 
and biochemical properties suggest a role in con- 
necting synaptic vesicles to each other and to the 
cytoskeleton in a regulated manner. It is hypothe- 
sized that the amino-terminal domain shared by all 
synapsins represents the active site for these bind- 
ing activities, which is differentially modulated in 
the four synapsins by the different carboxyl-termi- 
nal domains and by phosphorylation. Different syn- 
apses might have different characteristics depen- 
dent on the pattern of synapsins they express. 
Studies are under way to determine if changes in 
neurotransmitter release at a synapse are associated 
with changes in the expression of synapsins. The 
laboratory is also attempting to characterize the 
human genes for the four synapsins and to corre- 
late their structures with the binding activities of 
the synapsins to synaptic vesicles and to the 
cytoskeleton. 
Functionally, synaptic vesicles have an active pro- 
ton pump of the endomembrane type and neuro- 
transmitter uptake systems that depend on the 
transmembrane energy gradient established by the 
proton pump. The synaptic vesicle proton pump 
appears to be identical to that observed in clathrin- 
coated vesicles and chromaffin granules. The pri- 
mary structure of one of its subunits from bovine 
brain was determined and found to be highly ho- 
mologous to a subunit of the archibacterial proton 
ATPase, but not to the bacterial or mitochondrial 
F1,F0 proton ATPase, suggesting that the synaptic 
vesicle proton pump is similar to if not identical 
with a ubiquitous eukaryotic enzyme. This enzyme 
has evolved from a precursor shared with archi- 
bacteria but not with eubacteria. At this point the 
proton pump is the only identified synaptic vesicle 
protein that is not specific to synaptic vesicles in 
neurons but generally shared by intracellular or- 
ganelles. 
Continued 
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