Isolation of a putative vesicular intermediate in the cell-free transfer of membrane from transitional endoplasmic reticulum to the Golgi apparatus of etiolated seedlings of garden pea
1993
Hellgren, L. | Morre, D.J. | Sellden, G. | Sandelius, A.S.
Fractions enriched in endoplasmic reticulum were prepared from etiolated seedlings of garden pea (Pisum sativum). When incubated with ATP plus a cytosol fraction, some membrane elements present in the fractions responded by forming 50-70 nm buds and vesicles. These 50-70 nm buds and vesicles closely resembled the transition vesicles considered to function in situ in the transfer of membranes from the endoplasmic reticulum to the Golgi apparatus. Following incubation with ATP and cytosol, the endoplasmic reticulum fractions were fractionated by preparative free-flow, electrophoresis into fractions of different electrophoretic mobilities. The major fraction consisted of endoplasmic reticulum vesicles. The 50-70 nm vesicles were concentrated in a small shoulder fraction of lesser electrophoretic mobility. To label the vesicles, the endoplasmic reticulum fractions were first incubated with [3H] inositol to label the phosphatidylinositol by an exchange reaction requiring magnesium ions and cytidine nucleosides. Non-radioactive acceptor Golgi apparatus was prepared by, sucrose gradient flotation and immobilized on strips of nitrocellulose. With transition vesicles concentrated by preparative free-flow electrophoresis, transfer was more efficient than with transitional endoplasmic reticulum and independent of both ATP and cytosol. The findings demonstrate a population of vesicles derived from endoplasmic reticulum in a cell-free system from plants that may represent an intermediate compartment in membrane transfer between the endoplasmic reticulum and the Golgi apparatus.
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