ATP-dependent association between subunits of Clp protease in pea chloroplasts

Tami Halperin, Oren Ostersetzer, Zach Adam*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-γ-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-γ-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade β-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.

Original languageAmerican English
Pages (from-to)614-619
Number of pages6
JournalPlanta
Volume213
Issue number4
DOIs
StatePublished - 2001

Bibliographical note

Funding Information:
Acknowledgement This work was su pported in part by a grant from BARD, the US–Israel Binational Agricultural Research and Development Fund to Z.A.

Keywords

  • Chloroplast
  • Clp protease
  • Pisum sativum
  • Proteolysis

Fingerprint

Dive into the research topics of 'ATP-dependent association between subunits of Clp protease in pea chloroplasts'. Together they form a unique fingerprint.

Cite this