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Chapter 5 Sequestration Assays for Mammalian Autophagy

Seglen, Per O., Øverbye, Anders, Sætre, Frank·2009·Methods in Enzymology
DOI 10.1016/s0076-6879(08)03605-7
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Abstract

Macroautophagic activity is most directly and precisely measured by a cargo sequestration assay. Long-lived, cytosolic proteins that are degraded exclusively by the autophagic-lysosomal pathway, such as lactate dehydrogenase (LDH) are suitable as endogenous sequestration probes. Autophagic sequestration is measured as transfer of the protein from the soluble (cytosolic) to the sedimentable (organelle-containing) cell fraction, using leupeptin or other proteinase inhibitors to block inactivation and degradation of the protein inside autophagic vacuoles. A convenient separation method is electrodisruption of the cells, followed by sedimentation of the organelle fraction through a Nycodenz density cushion. A promising variant of the cargo assay is to use a protein probe that is processed by the autophagic-lysosomal pathway so as to generate an intravacuolar fragment. Because there is no cytosolic background, subcellular fractionation is unnecessary, allowing the use of the autophagic fragment assay to measure autophagic activity in whole cells. In hepatocytes, a small fragment, p10(BHMT), made by autophagic processing of the enzyme betaine:homocysteine methyltransferase, thus accumulates in an autophagy-dependent manner in the presence of leupeptin. Autophagic sequestration can also be measured by using exogenous cargo probes, such as radiolabeled di- and trisaccharides, which can be loaded into the cytosol of hepatocytes by reversible electrodisruption or mechanical stress. Raf…

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Metadata

Authors
Seglen, P. O., Øverbye, A., & Sætre, F.
Venue
Methods in Enzymology
Publisher
Elsevier
Published
2009
Pages
63-83
ISSN
0076-6879
DOI
10.1016/s0076-6879(08)03605-7
Cited by
18
References
8
Language
en

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Seglen, P. O., Øverbye, A., & Sætre, F. (2009). Chapter 5 Sequestration Assays for Mammalian Autophagy. Methods in Enzymology. https://doi.org/10.1016/s0076-6879(08)03605-7