Coexistence of Group I and Group II Chaperonins in the Archaeon Methanosarcina mazei

Daniel Klunker,Bernd Haas,Angela Hirtreiter,Luis Figueiredo,Dean J Naylor,Günter Pfeifer,Volker Müller,Uwe Deppenmeier,Gerhard Gottschalk,F.Ulrich Hartl,Manajit Hayer-Hartl

doi:10.1074/jbc.m302018200

Abstract

Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Gö1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.

Highlights

The chaperonins are a structurally conserved class of molecular chaperones that assist, in an ATP-dependent manner, in the efficient folding of a subset of newly synthesized and stressdenatured polypeptide chains [1,2,3,4,5,6,7,8]
Chaperonin Genes in the M. mazei Go1 Genome—Analysis of the genome of the mesophilic archaeon M. mazei Go1 (Mm) revealed the existence of three conserved genes coding for Ths subunits
We identified two genes in the M. mazei genome encoding the subunits of the potential Ths cofactor Pfd, which is known to assist in group II chaperonin-mediated protein folding in the eukaryotic cytosol [27,28,29,30,31, 55]

Summary

The abbreviations used are

Chaperonin; CCT, chaperonin containing TCP-1; DTT, dithiothreitol; Ec, E. coli; hs, H. sapiens; HSE, heat shock element; Hsp, heat shock protein; IPTG, isopropyl-1-thio-␤D-thiogalactopyranoside; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; ␤-ME, ␤-mercaptoethanol; Mm, M. mazei; MOPS, 4-morpholinepropanesulfonic acid; OD, optical density; Pfd, prefoldin; Ta, T. acidophilum; Ths, thermosome; TRiC, TCP-1 containing ring complex. This report describes the molecular cloning, purification, reconstitution, and preliminary functional analysis of both types of chaperonin from M. mazei Go1 We demonstrate that both chaperonins are expressed under standard growth conditions of M. mazei and coexist in the cytosolic compartment at a ratio of GroEL to Ths of ϳ1:1. Both chaperonins are capable of preventing the aggregation of denatured model substrate proteins, such as mitochondrial rhodanese, but only the GroEL/GroES system supports rhodanese refolding

EXPERIMENTAL PROCEDURES

Miscellaneous Procedures

RESULTS

DISCUSSION