MEMBRANE INSERTION AND INTRACELLULAR-TRANSPORT OF YEAST SYNTAXIN SSO2P IN MAMMALIAN-CELLS

Proteins of the syntaxin family are suggested to play a key role in de termining the specificity of intracellular membrane fusion events. The y belong to the class of membrane proteins which are devoid of N-termi nal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 ce lls using the Semliki Forest virus vector, and its localization and mo de of membrane insertion were studied. By immunofluorescence and immun o-EM we show that Sso2p is transported to its final location, the plas ma membrane, along the biosynthetic pathway. Experiments with synchron ized Sso2p synthesis or expression of the protein in the presence of b refeldin A indicate endoplasmic reticulum as the initial membrane inse rtion site. During a 20 degrees C temperature block Sso2p, accumulated in the Golgi complex and was chased to the plasma membrane by a subse quent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic mi crosomes posttranslationally. A truncated form of Sso2p lacking the pu tative membrane anchor was used to show that this sequence is necessar y for the membrane insertion in vivo and in vitro. The results show th at this syntaxin-like protein does not directly associate with its tar get membrane but uses the secretory pathway to reach its cellular loca tion, raising interesting questions concerning regulation of SNARE-typ e protein function.