The Presence of Ubiquitin-Protein Conjugates in Plant Chloroplast Lysates Is Due to Cytosolic Contamination

Abstract

Chloroplasts were isolated by Percoll density gradient centrifugation from Lemna minor and Spinacia oleracea grown under normal conditions or from L. minor subjected to sulfur starvation, a condition that induces a dramatic increase in the rates of proteolysis, namely in the degradation of the chloroplast enzyme ribulosebisphosphate carboxylase. The presence of free ubiquitin or ubiquitin-protein conjugates was not immunologically detected in the purified chloroplast extracts under either of these conditions. Moreover, no ATP-dependent conjugation of ¹²⁵I-labelled ubiquitin to endogenous proteins was detected in L. minor chloroplast lysates. Our results support the view that plant chloroplasts do not possess free ubiquitin or ubiquitin conjugating activity. Furthermore, treatment of the mechanically isolated chloroplasts with thermolysin suggests that the presence of ubiquitin-protein conjugates in some chloroplast preparations is due to cytosolic contamination.