FRAGMENTATION OF A GOLGI-LOCALIZED CHIMERIC PROTEIN ALLOWS DETERGENT SOLUBILIZATION AND REVEALS AN ALTERNATE CONFORMATION OF THE CYTOPLASMIC DOMAIN

Golgi resident proteins maintain their localization despite a continua l protein and lipid flux through the organelle. To study Golgi retenti on mechanisms, we have focused upon the chimeric protein Gml. This pro tein contains the Golgi transmembrane domain targeting signal from the infectious bronchitis virus M protein and the lumenal and cytoplasmic domain of the vesicular stomatitis virus glycoprotein (VSV G). The Gm l protein is targeted to the Golgi where it forms an unusually stable detergent-resistant oligomer. The formation of oligomeric structures m ay aid retention of Golgi resident proteins. Thus, determining the sta bilization mechanism may shed light on Golgi protein retention. Previo us work determined that the transmembrane domain is required for the t argeting and oligomerization of Gml, but it is the cytoplasmic tail th at stabilizes the complexes [Weisz, O. A., Swift, A. M., and Machamer, C. E. (1993) J. Cell Biol. 122, 1185-1196]. However, further study of the oligomer has been difficult due to its insolubility. Here we repo rt that fragmenting the Gml protein into several pieces facilitates so lubilization by sodium dodecyl sulfate (SDS). By analyzing the fragmen ts produced after cleavage, we determined that the stability of the ol igomer is not caused by covalent linkage of Gml to itself or other pro teins. The fragment corresponding to the transmembrane domain and tail of Gml had an enhanced mobility in SDS gels relative to the same frag ment of the parent VSV G protein, The enhanced migration of the tail f ragment does not reflect sequence differences or post-translational mo dification, but correlates with Golgi localization and oligomerization . We suggest that the enhanced mobility of the Gml tail fragment refle cts an altered conformation which serves to stabilize the detergent-re sistant oligomers.