Use of a Phosphatidylinositol Phosphate Affinity Chromatography (PIP Chromatography) for the Isolation of Proteins Involved in Protein Quality Control and Proteostasis Mechanisms in Plants

Methods Mol Biol. 2016;1450:223-32. doi: 10.1007/978-1-4939-3759-2_18.

Abstract

Protein functionality depends directly on its accurately defined three-dimensional organization, correct and efficient posttranslational modification, and transport. However, proteins are continuously under a hostile environment threatening with folding aberrations, aggregation, and mistargeting. Therefore, proteins must be constantly "followed up" by a tightly regulated homeostatic mechanism specifically known as proteostasis. To this end other proteins ensure this close surveillance including chaperones as well as structural and functional members of the proteolytic mechanisms, mainly the autophagy and the proteasome related. They accomplish their action via interactions not only with other proteins but also with lipids as well as cytoskeletal components. We describe a protocol based on an affinity chromatographic approach aiming at the isolation of phosphatidyl inositol phosphate binding proteins, a procedure which results into the enrichment and purification of several members of the proteostasis mechanism, e.g. autophagy and proteasome, among other components of the cell signaling pathways.

Keywords: Autophagy; Chaperones; Phosphatidylinositol phosphate (PIP); Proteasome; Sumoylation; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / genetics
  • Chromatography, Affinity / methods*
  • Molecular Biology / methods*
  • Phosphatidylinositol Phosphates / chemistry
  • Phosphatidylinositol Phosphates / genetics
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification*
  • Protein Aggregates / genetics*
  • Protein Folding
  • Proteostasis / genetics

Substances

  • Phosphatidylinositol Phosphates
  • Plant Proteins
  • Protein Aggregates