Identification of the principal ATPase associated with transitional endoplasmic reticulum (TER) of rat liver

Lin Zhang, Purdue University

Abstract

The transfer of membrane from the endoplasmic reticulum (ER) to the Golgi apparatus occurs via 50-70 nm diameter transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE isolated from rat liver TER and designated as TER ATPase is a hexamer of six 100 kD subunits with a native molecular weight of 600 kD. The 100 kD protein hydrolyzes ($\gamma$-$\sp{32}$P) ATP and is phosphorylated in the presence of Mg$\sp{2+}.$ It is distinct from the classical transport ATPase based on pH optima, ion effects and inhibitor specificity. Electron microscopy of negatively stained preparations revealed TER ATPase as a ring-shaped structure with a six-fold rotational symmetry. A sequenced 19 amino acid peptide of the TER ATPase has 84% identity with valosin-containing protein (VCP) and 64% identity with a yeast cell-cycle control protein cdc48p. Anti-synthetic peptide antisera to a 15 amino acid portion of the sequence of TER ATPase recognizes a 100 kD protein from TER. These antisera reduces the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibit transfer of radiolabeled material from ER to Golgi apparatus, while preimmune sera do not. The results suggest that TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase obtained by immunoscreening a rat liver CDNA library with the affinity-purified TER ATPase antibody reveal TER ATPase to be the rat equivalent of porcine valosin-containing protein. It is a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide sensitive fusion protein.

Degree

Ph.D.

Advisors

Morre, Purdue University.

Subject Area

Biology|Biochemistry|Cellular biology

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