ISOLATION AND CHARACTERIZATION OF THE PRINCIPAL ATPASE ASSOCIATED WITH TRANSITIONAL ENDOPLASMIC-RETICULUM OF RAT-LIVER

Citation
L. Zhang et al., ISOLATION AND CHARACTERIZATION OF THE PRINCIPAL ATPASE ASSOCIATED WITH TRANSITIONAL ENDOPLASMIC-RETICULUM OF RAT-LIVER, The Journal of cell biology, 127(6), 1994, pp. 1871-1883
Citations number
54
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
127
Issue
6
Year of publication
1994
Part
2
Pages
1871 - 1883
Database
ISI
SICI code
0021-9525(1994)127:6<1871:IACOTP>2.0.ZU;2-L
Abstract
The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from pa rt rough, part-smooth transitional elements of the endoplasmic reticul um (TER). Vesicle budding from the TER is an ATP-dependent process bot h in vivo and in vitro. An ATPase with a monomer molecular weight of 1 00 kD by SDS-PAGE has been isolated from TER and designated as TER ATP ase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysi s of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negativ ely stained preparations revealed the TER ATPase to be a ring-shaped s tructure with sixfold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64 % identity with a yeast cell-cycle control protein CDC48p was obtained . Anti-synthetic peptide antisera to a 15-amino acid portion of the se quence of TER ATPase recognized a 100-kD protein from TER. These antis era reduced the ATP-dependent cell-free formation of transition vesicl es from isolated TER of rat liver. In a reconstituted membrane transfe r system, TER ATPase antisera inhibited transfer of radiolabeled mater ial from endoplasmic reticulum to Golgi apparatus, while preimmune ser a did not. The results suggest that the TER ATPase is obligatorily inv olved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreen ing a rat liver cDNA library with the affinity-purified TER ATPase ant ibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containi ng protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.