GIANTIN, A NOVEL CONSERVED GOLGI MEMBRANE-PROTEIN CONTAINING A CYTOPLASMIC DOMAIN OF AT LEAST 350-KDA

The Golgi complex consists of a series of stacked cisternae in most eu karyotes. Morphological studies indicate the existence of intercistern al cross-bridge structures that may mediate stacking, but their identi ty is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofl uorescence experiments was coincident with that of galactosyltransfera se, both in untreated cells and in cells treated with agents that disr upt Golgi structure. A monoclonal antibody against giantin yielded Gol gi staining in one avian and all mammalian cell types tested, indicati ng that giantin is a conserved protein. Giantin exhibited reduced mobi lity on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrop horesis, was recovered in membrane fractions after differential centri fugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral componen t of the Golgi membrane with a disulfide-linked lumenal domain. Striki ngly, the majority of the polypeptide chain is cytoplasmically dispose d, because large (up to 350 kDa) proteolytic fragments of giantin coul d be released from intact Golgi vesicles. This feature, a large contig uous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tu bule membranes. Therefore, giantin's localization, conservation, and p hysical properties suggest that it may participate in forming the inte rcisternal cross-bridges of the Golgi complex.