Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa.

Abstract

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.

Two Mammalian Sec16 Homologues Have Nonredundant Functions in Endoplasmic Reticulum (ER) Export and Transitional ER Organization

“…Although Sec23A was still detectable in the juxtanuclear Golgi region, the punctate pattern was replaced with more homogeneous staining. In the same cells, we visualized the Golgi with an antibody against the membrane-anchored coiled-coil protein giantin (Linstedt and Hauri, 1993). Treatment with RNAi against Sec16L or Sec16S did not substantially alter the juxtanuclear Golgi staining ( Figure 3B).…”

GRASP55 Regulates Golgi Ribbon Formation

“…Primary antibodies used were as follows: anti-Myc 9e10 (Jesch et al, 2001), anti-giantin (Linstedt and Hauri, 1993), anti-p115 (Puthenveedu and Linstedt, 2001), anti-GM130 (Puthenveedu and Linstedt, 2001), anti-GP73 (Bachert et al, 2007), anti-GPP130 (Linstedt et al, 1997), and anti-GRASP55, which was generated by immunizing rabbits (Covance, Emeryville, CA) with purified glutathione transferase (GST)-GRASP55 (Jesch et al, 2001). Affinity-purified, conjugated secondary antibodies were purchased (Invitrogen, Carlsbad, CA).…”

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…Primary antibodies used were as follows: anti-ERK2 C16 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GPP130 (Bachert et al, 2001), anti-p115 (Puthenveedu and Linstedt, 2001a), anti-GRASP65 (Wang et al, 2003), diphospho-specific anti-ERK1/2 (Santa Cruz Biotechnology), anti-hemagglutinin (HA) (Bachert et al, 2001), anti-Myc 9e10 (Jesch et al, 2001a), and anti-giantin (Linstedt and Hauri, 1993). Secondary antibodies were affinity purified and fluorescein isothiocyanate or rhodamine isothiocyanate conjugated (Invitrogen, Carlsbad, CA).…”