Fragmentation of a Golgi-Localized Chimeric Protein Allows Detergent Solubilization and Reveals an Alternate Conformation of the Cytoplasmic Domain

Abstract

Golgi resident proteins maintain their localization despite a continual protein and lipid flux through the organelle. To study Golgi retention mechanisms, we have focused upon the chimeric protein Gm1. This protein 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 Gm1 protein is targeted to the Golgi where it forms an unusually stable detergent-resistant oligomer. The formation of oligomeric structures may aid retention of Golgi resident proteins. Thus, determining the stabilization mechanism may shed light on Golgi protein retention. Previous work determined that the transmembrane domain is required for the targeting and oligomerization of Gm1, but it is the cytoplasmic tail that 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 report that fragmenting the Gm1 protein into several pieces facilitates solubilization by sodium dodecyl sulfate (SDS). By analyzing the fragments produced after cleavage, we determined that the stability of the oligomer is not caused by covalent linkage of Gm1 to itself or other proteins. The fragment corresponding to the transmembrane domain and tail of Gm1 had an enhanced mobility in SDS gels relative to the same fragment of the parent VSV G protein. The enhanced migration of the tail fragment does not reflect sequence differences or post-translational modification, but correlates with Golgi localization and oligomerization. We suggest that the enhanced mobility of the Gm1 tail fragment reflects an altered conformation which serves to stabilize the detergent-resistant oligomers.

Cerebroside Sulfotransferase Forms Homodimers in Living Cells

“…Whether dimerization of CST is required for ER export remains to be determined because it is not possible to ascertain whether the CST(1-36)EGFP protein completely mimics the trafficking of the native CST protein. Similarly, hetero-oligomerization, which might be mediated by specific residues in the lumenal domain (28), has been suggested to be responsible for Golgi retention of glycosyltransferases (16). In addition, we were able to show that monomeric CST-(1-36)EGFP and CST(1-63)EGFP proteins were exclusively localized to the Golgi apparatus.…”

Expression and Purification of N and E Proteins from Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus: a Comparative Study

“…These characteristics might account for its strong propensity to associate with the membrane. Albeit uncommon, such oligomer could have formed detergent-resistant structures (Weisz et al 1993, Sevier & Machamer 1998, so that the detergents commonly used for solubilizing integral membrane proteins failed to completely solubilize rEH. In this study, we have tested parameters such as salt concentrations (0.15--1 M NaCl), sonication time (0.5--1.5 min per 0.5 ml solution), incubation time (30 min to overnight) (data not shown) and detergent types (see Figure 4) in order to extract rEH from the pellets more efficiently, but only a limited degree of success was achieved.…”

Oligomerization and Topology of the Golgi Membrane Protein Glucosylceramide Synthase

“…The oligomerization of Golgi membrane proteins has been suggested to be involved in their retention in the Golgi complex (25)(26)(27)(28)(29). In addition, several other glycosyltransferases have been shown to have a dimeric or oligomeric structure, suggesting a functional role of oligomerization in such enzymes (30 -33).…”