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 ~ 310 kD, probably homohexamers. The protein has a transmembrane topology with only a short cytoplasmic segment as assessed by protease protection experiments.
A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [3SS]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.
Protein traffic moving from the endoplasmic reticulum (ER) to the Golgi complex in mammalian cells passes through the tubulovesicular membrane clusters of the ER-Golgi intermediate compartment (ERGIC), the marker of which is the lectin ERGIC-53. The dynamic nature and functional role of the ERGIC have been debated for quite some time. In the most popular current view, the ERGIC clusters are mobile transport complexes that deliver secretory cargo from ER-exit sites to the Golgi. Recent live-cell imaging data revealing the formation of anterograde carriers from stationary ERGIC-53-positive membranes, however, suggest a stable compartment model in which ER-derived cargo is first shuttled from ER-exit sites to stationary ERGIC clusters in a COPII-dependent step and subsequently to the Golgi in a second vesicular transport step. This model can better accommodate previous morphological and functional data on ER-to-Golgi traffic. Such a stationary ERGIC would be a major site of anterograde and retrograde sorting that is controlled by coat proteins, Rab and Arf GTPases, as well as tethering complexes, SNAREs and cytoskeletal networks. The ERGIC also contributes to the concentration, folding, and quality control of newly synthesized proteins.
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