THE 60 KDA HEAT-SHOCK PROTEINS IN THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS-SHIBATAE

One of the most abundant proteins in the hyperthermophilic archaeon Su lfolobus shibatae is the 59 kDa heat shock protein (TF55) that is beli eved to form a homo-oligomeric double ring complex structurally simila r to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoich iometric with TF55 (renamed beta). The gene and flanking regions of al pha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start site s for both alpha and beta were mapped and three potential transcriptio n regulatory regions were identified. Northern analyses of cultures sh ifted from normal growth temperatures (70 to 75 degrees C) to heat sho ck temperatures (85 to 90 degrees C) indicated that the levels of alph a and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synth esis by autoradiography of total proteins from cultures pulse labeled with L-[S-35]methionine at normal and heat shock temperatures indicate d significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (greater than or equal to 90 degr ees C) alpha and beta appeared to be the only two proteins synthesized . The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of th e two subunits gave the greatest yield of the complex, which we refer to as a ''rosettasome''. It is argued that the rosettasome consists of two homo oligomeric rings; one of alpha and the other of beta. Polycl onal antibodies against alpha and beta from S, shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal s pecies tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettas omes and the eukaryotic TCP1 protein family and the distant relationsh ip to the bacterial GroEL/HSP60 proteins. (C) 1995 Academic Press Limi ted