Intracellular Localization of Heat Shock Proteins in Maize

Abstract

The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centiftion was employed to separate organeles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacryl-amide gel ekectrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelie associated. Hsp 25 and 72 were in fractions contining closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fraction containg overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better sepation of the two sets ofclosely equilibrating organelles. When a 13,000g centrifigtion step to remove mitochondria was employed prior to gradient centrifngation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either aintaining the ribosomes on the membrne or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments. A universal response to hyperthermic temperatures in a diversity of organisms is the synthesis of hsp3 (for recent review, see Ref. 21). We have previously characterized this heat shock response in maize (6, 7). Although ubiquitin (3), a poly(A)-binding protein (29), and an ATP-dependent protease (12) have been identified as heat indu-cible proteins, the identity and function of most of the hsp remains unclear. However, several lines of evidence point to the proteins' role in protecting organisms from the lethal effects of extremely high temperatures. The kinetics of thermotolerance induction and decay correlate with the kinetics of hsp synthesis and degradation in numerous organisms (21) including higher plants (20). Analysis of mutants also demonstrates a relationship between the ability of an organism to survive high temperature extremes and the synthesis of hsp. In Escherichia coli. mutants for the regulatory hin gene cannot synthesize hsp and die at high temperatures (36). A Dictyostelium mutant incapable ofsynthesizing the small hsp is unable to acquire thermotolerance (22). Double mutations in hsp 70 genes confer temperature sensitive growth