Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus.

TLDR

Purified Golgi membranes from Caco‑2 cells were used to generate the monoclonal antibody G1/93, which specifically labels a tubulovesicular compartment at the cis‑side of the Golgi apparatus, including the first cis‑cisterna, as visualized by immunoelectron microscopy with galactosyltransferase antibodies. The G1/93 antigen is a 53‑kDa transmembrane protein that dimerizes and oligomerizes into ~310‑kDa homohexamers immediately after synthesis, is likely non‑glycosylated, and its specific localization to the cis‑Golgi defines the ER‑to‑Golgi transport route, making G1/93 a useful cis‑Golgi marker in human cells.

Abstract

Purified Golgi membranes of the human intestinal adenocarcinoma cell line Caco-2 were used as an antigen to produce a monoclonal antibody, G1/93, which specifically labels a tubulovesicular compartment near the cis side of the Golgi apparatus, including the first cis-cisterna itself, as visualized by single and double immunoelectron microscopy with antibodies against galactosyltransferase. The antigen recognized by G1/93 was identified as a protein with a subunit size of 53 kD. Pulse-chase experiments revealed that the 53-kD protein dimerizes immediately after synthesis followed by formation of oligomers of approximately 310 kD, probably homohexamers. The protein has a transmembrane topology with only a short cytoplasmic segment as assessed by protease protection experiments. Glycosidase digestion studies indicated that the protein is probably not glycosylated. The unique subcellular distribution of the G1/93 antigen in close vicinity to the cis-Golgi is in line with the notion that this protein may delineate the biosynthetic transport pathway from the endoplasmic reticulum to the Golgi apparatus. Moreover, G1/93 is a useful marker to identify the cis side of the Golgi apparatus in a variety of human cells.