The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis-Golgi.
A procedure has been established in Vero cells for the isolation of an intermediate compartment involved in protein transport from the ER to the Golgi apparatus. The two-step subcellular fractionation procedure consists of Percoll followed by Metrizamide gradient centrifugation. Using the previously characterized p53 as a marker protein, the average enrichment factor of the intermediate compartment was 41. The purified fraction displayed a unique polypeptide pattern. It was largely separated from the rough ER proteins ribophorin I, ribophorin II, BIP, and protein disulfide isomerase, as well as from the putative cis-Golgi marker N-acetylglucosamine-1-phosphodiester-alpha-N-acetylglucosaminidase, the second of the two enzymes generating the lysosomal targeting signal mannose-6-phosphate. The first enzyme, N-acetylglucosaminylphosphotransferase, for which previous biochemical evidence had suggested both a pre- and a cis-Golgi localization in other cell types, cofractionated with the cis-Golgi rather than the intermediate compartment in Vero cells. The results suggest that the intermediate compartment defined by p53 has unique properties and does not exhibit typical features of rough ER and cis-Golgi.
Schweizer
A
A
Department of Pharmacology, Biocenter, University of Basel, Switzerland.
Matter
K
K
Ketcham
C M
CM
Hauri
H P
HP
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure*; D006056:Golgi Apparatus / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D014709:Vero Cells
D008565:Membrane Proteins