MOLECULAR-GENETIC ANALYSES OF A 376-KILODALTON GOLGI-COMPLEX MEMBRANE-PROTEIN (GIANTIN)

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane pr otein (giantin) are described. The immunoglobulin G fraction of a huma n serum containing antibodies against GC antigens as revealed by indir ect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybrid izing clones. Additional cDNA clones were retrieved from a lambdagt11 human thyroid cDNA library or generated by reverse transcriptase-media ted PCR from HeLa cell mRNA. Alignment of the clones resulted in a con sensus cDNA of 10,300 bp encoding a protein of 376 kDa. The correspond ing mRNA with a size of about 10 kb was detected by Northern (RNA) blo tting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the p rotein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of th e myosin family. Five overlapping recombinant proteins covering the en tire sequence were synthesized and used for antibody production in rab bits and for affinity purification of human and rabbit antibodies. Ind irect immunofluorescence experiments also done with brefeldin A-treate d Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experim ents with antibodies against the GC marker mannosidase II as well as i mmunoelectron microscopic studies confirmed the localization of the pr otein within the GC. A corresponding endogenous large-molecular-mass p rotein of about 390 kDa was found in [S-35]methionine-labeled Hep-2 ce ll lysates as well as in GC-enriched subcellular fractions from rat li ver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functi ons in scaffold formation or vesicle transport. As far as can be concl uded from immunological and personally communicated partial cDNA seque nce data, the protein seems to be identical with a 400-kDa Golgi prote in (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name gi antin.