Heat shock enhances the amount of prenylated Dnaj protein at membranes of glyoxysomes

Eur J Biochem. 1994 Jan 15;219(1-2):57-63. doi: 10.1111/j.1432-1033.1994.tb19914.x.

Abstract

Proteins similar to the bacterial Dnaj protein have been implicated as molecular chaperones in different compartments of eukaryots. A plant equivalent is now described in tissues of dark-grown cucumber seedlings. Using a cucumber Dnaj protein produced by expression in bacteria, we raised polyclonal antibodies against the protein and used them for localization studies. In etiolated cucumber seedlings, both cotyledons and hypocotyledons were found to contain Dnaj proteins. Cell fractionation of etiolated cotyledons showed that Dnaj proteins were detectable mainly in the postnuclear cell fraction after sedimentation at 10,000 x g, and in the microsomes. Following subfractionation by sucrose density gradient centrifugation and analysis by immunoblotting, a 53-kDa protein was attributed to the glyoxysomal fraction and an 80-kDa protein to the mitochondrial fraction. The glyoxysomal Dnaj protein behaved as a membrane-bound form. Upon heat shock, a slight increase in the content of the glyoxysomal Dnaj protein was found. When glyoxysomes were treated with protease and subsequently isolated by gradient centrifugation, virtually all immunologically detectable Dnaj protein was removed. Administration of radiolabelled mevalonic acid to cotyledons and isolation of glyoxysomes yielded labelled Dnaj protein which remained membrane bound during the purification of glyoxysomal membranes by floatation in a density gradient.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation
  • Centrifugation, Density Gradient
  • Endopeptidases / metabolism
  • Gene Library
  • HSP40 Heat-Shock Proteins
  • Heat-Shock Proteins / biosynthesis*
  • Heat-Shock Proteins / isolation & purification
  • Hot Temperature
  • Intracellular Membranes / metabolism*
  • Membrane Proteins / biosynthesis*
  • Membrane Proteins / isolation & purification
  • Mevalonic Acid / metabolism
  • Molecular Weight
  • Organelles / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Seeds / metabolism
  • Subcellular Fractions / metabolism
  • Tritium
  • Vegetables / metabolism*

Substances

  • HSP40 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Membrane Proteins
  • Recombinant Proteins
  • Tritium
  • Endopeptidases
  • Mevalonic Acid