Abstract
Palmitoylation is a post translational modification of proteins which binds palmitic acid
molecules (C16:0) covalently via cycteine residues to the polypeptide chain. This kind
of modification has been reported for many viral and cellular proteins but structural
and functional aspects of this fatty acid attachment could not be completely clarified
yet.
The aim of this work was to examine the molecular requirements for the binding of
palmitic acid to cystein residues of the vesicular membrane protein synaptotagmin I.
The consequences of the loss of these fatty acids for intracellular processing and
function of this protein were also investigated. To resolve these questions mutants of
the synaptotagmin I gene which lack either one or more cystein within their putative
palmitoylation region were constructed. These constructs were expressed in several
cell lines using the vaccinia T7 system or by transfection with cationic lipids (lipofectin).
The expressed proteins were characterized either by metabolic labelling or by
detection with antibodies coupled with a fluorescence dye. The results can be summarized
as follows:
A) All five cysteins within the palmitoylation region of synaptotagmin I are
either directly or indirectly involved in the attachment of palmitic acid. Only the total
mutant in which all cysteins were substituted by serines showed no incorporation of (3)H-labelled palmitic acid. A selective substitution of certain cysteines to serins reduced
the rate of incorporation to about 40% of the rate found for wild type synaptotagmin.
B) In CV1 and BON cells the expressed synaptotagmin showed a characteristic
pattern of glycosylation products. Pulse chase experiments revealed that the
highly glycosylated and presumably physiologically active form of the protein does
not appear in the cells until two hours after labelling. The fatty acid free mutant
showed a significantly retarded maturation. Even after two hour of incubation the mutant
does not reach the highly glycosylated form.
C) Cell fractionation experiments confirmed that the lack of palmitoylation
and/or the substitution of the cysteins affected the transport of the newly synthesized
protein. In differentiated PC12 cells fluorescence microscopic investigation of transport
of the wild-type protein and the fatty acid free mutant resulted in no differences in
intracellular localisation after long term expression. The targeting of both expression
products to the axon terminals seemed normal.
The results of the experiments show that fatty acids covalently attached to the polypeptide
chain of synaptotagmin I are important for certain steps in the transport pathway
of the protein although no complete block of transport events occured if acylation
was prevented. No distinct function of this modification was found yet. | 
