Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo.
Using Agrobacterium, we developed a method to transform an Arabidopsis cell suspension culture. A stably transformed cell line expressing high levels of firefly luciferase (Luc) was used for in vivo studies of thermal denaturation and renaturation of the enzyme and the protective role of different chaperones. Luc activity was monitored under heat stress and recovery conditions in control, thermotolerant cells and cells expressing plant chaperones after transient cotransformation with plasmids encoding proteins of the heat shock protein Hsp90, Hsp70, or Hsp20 family. The effects of the expressed proteins were specific. The Hsp17.6 class I protein maintained Luc activity on a level comparable with that observed in thermotolerant cells and improved Luc renaturation. Although transient expression of Hsp90 did not protect Luc from thermal denaturation, it accelerated Luc renaturation during recovery. In contrast to the other chaperones tested, overexpression of Hsp70 alone had no effect on denaturation and renaturation of Luc but enhanced Luc renaturation if coexpressed with Hsp17.6.
Forreiter
C
C
Department of Molecular Cell Biology, Goethe University, Frankfurt am Main, Germany. forreiter@cellbiology.uni-frankfurt.de
Kirschner
M
M
Nover
L
L
D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000502:physiology*; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000502:physiology*; D011489:Protein Denaturation; D012231:Rhizobium / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D008156:Luciferases