LOCALIZATION OF SED5, A PUTATIVE VESICLE TARGETING MOLECULE, TO THE CIS-GOLGI NETWORK INVOLVES BOTH ITS TRANSMEMBRANE AND CYTOPLASMIC DOMAINS

The yeast Sed5 protein, which is required for vesicular transport betw een ER and Golgi complex, is a membrane protein of the syntaxin family . These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism b y which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cell s results in a perinuclear distribution; immuno-EM reveals that the ma jority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane -spanning domain contains targeting information, as is the case with r esident Golgi enzymes. However, alterations to the transmembrane domai n of Drosophila Sed5 itself did not result in its mistargeting, implyi ng that an additional targeting mechanism exists which involves only t he cytoplasmic part of the protein. This was confirmed by modifying th e transmembrane domain of the yeast Sed5 protein: substitution with th e corresponding region from the Sso1 protein (a plasma membrane syntax in homologue) did not affect yeast Sed5 function in vivo.