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

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 lOOOOXg, 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 mitochondria1 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.The knowledge about molecular chaperones participating in the unidirectional intracellular transport of proteins had great implications in formulating concepts of organelle biosynthesis [l-31. However, new concepts and approaches are required, particularly for improving our understanding of the biosynthesis of peroxisomes. Despite the elegant work with in-vivo-expressed chimeric proteins and the characterization of peroxisomal-targeting sequences [4], the studies needed to understand the import machinery were only partly successful [5-81. Hence, genetic approaches have been applied [9, 101 to overcome inadequacies in known methodologies. Also promising are investigations of peroxisomal membrane components which are responsible for the specific binding of cytosolic precursor proteins destined for the peroxisomal matrix. It is anticipated that the peroxisomal location signal itself or a potential cytosolic receptor, a peroxisomal location signal-binding protein, may be involved in selecting the precursor protein from the cytosolic pool.Cells programmed for increased peroxisome or glyoxysome biosynthesis are assumed to promote substantial production of components of the peroxisomal-import machinery. To detect components of this apparatus, we began with a cDNA library representing the stage just prior to the stage of maximal fatty acid degradation. This metabolic process is preceded by a stage of extensive synthesis and import ofCorrespondence to H. Kindl,

The Effects of Chaperones and the Influence of Protein Assembly on Peroxisomal Protein Import

“…Chaperones Hsp70 and Hsp90 were among the proteins identified whose expression is up-regulated during the heat shock response (39). Hsp70 and Hsp40 have been localized to peroxisomal membranes (27)(28)(29), and the amount of Hsp40 associated with these membranes increases after heat stress (28). An Hsp70 has also recently been identified in the matrix of watermelon glyoxysomes (50).…”

“…Many researchers have reported that the folding state of peroxisomal proteins is not critical for membrane translocation (30 -34). On the other hand, chaperones, which are known to control protein folding reactions, have been implicated in peroxisomal protein import (27)(28)(29). To resolve this apparent conflict, we examined the participation of Hsps in protein import, the interactions between cytoplasmic Hsps and peroxisomal proteins, and the effect of the assembly state of the targeted protein on import efficiency.…”

“…An Hsp70 was recently identified in the matrix of watermelon glyoxysomes (50). In addition, levels of prenylated, glyoxysomal membrane-associated DnaJ (Hsp40) are enhanced with heat shock (28). Further experiments will be needed to demonstrate a direct role for any of these factors during peroxisomal protein import.…”

“…In addition, an Hsp70 colocalizes with PTS-containing proteins and with peroxisomal membranes, and antibodies directed against bovine cytosolic Hsp73 inhibit peroxisomal protein import (27). Finally, an Hsp40 and an Hsp70 have been localized to the glyoxysomal membrane (28,29); an Hsp70 has recently been identified in the matrix of glyoxysomes (50).…”

The Hsp70 and Hsp40 Chaperones Influence Microtubule Stability inChlamydomonas

“…Alignment of the Chlamydomonas protein with the plant proteins shows that it contains an N-terminal extension of 19 amino acids enriched in methionine, glycine, phenylalanine, and proline. Like other Hsp40 proteins from plants, the Chlamydomonas Dnj1 protein contains a CAQQ motif at the C terminus (Frugis et al 1999;Nambara and McCourt 1999), suggesting that the protein may be post-translationally modified by prenylation as has been demonstrated for some plant DnaJ proteins (Preisig-Muller et al 1994) and for the S. cerevisiae Ydj1 protein (Caplan et al 1992). …”