[1] Membrane isolation: Strategy, techniques, markers

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.

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Plasma Membrane Isolation by Phase Partition using a Single Partition Step: A Tool for the in vivo Study of Lipid Transfer to the Plasma Membrane

Separations Using Aqueous Phase Systems ◽

10.1007/978-1-4684-5667-7_10 ◽

1989 ◽

pp. 51-58

Author(s):

Patrick Moreau ◽

Hélène Juguelin ◽

René Lessire ◽

Claude Cassagne

Keyword(s):

Plasma Membrane ◽

In Vivo Study ◽

Lipid Transfer ◽

Phase Partition ◽

Membrane Isolation ◽

Plasma Membrane Isolation

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Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

The Journal of Cell Biology ◽

10.1083/jcb.75.1.119 ◽

1977 ◽

Vol 75 (1) ◽

pp. 119-134 ◽

Cited By ~ 86

Author(s):

C M Cohen ◽

D I Kalish ◽

B S Jacobson ◽

D Branton

Keyword(s):

Adenosine Triphosphatase ◽

Wheat Germ ◽

Plasma Membranes ◽

Cell Attachment ◽

Isolation Procedure ◽

Chemical Analyses ◽

Isolation Techniques ◽

Cell Plasma ◽

Membrane Bound ◽

Membrane Isolation

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

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