HEAT-SHOCK-PROTEIN-70 FAMILY - MULTIPLE SEQUENCE COMPARISONS, FUNCTION, AND EVOLUTION

The heat shook protein 70 kDa sequences (HSP70) are of great importanc e as molecular chaperones in protein folding and transport. They are a bundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organell es (mitochondria, chloroplasts), A multiple alignment of a large colle ction of these sequences was obtained employing our symmetric-iterativ e ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conse rvation are interpreted in evolutionary terms and with respect to func tional implications. Many archaeal sequences (methanogens and halophil es) tend to align best with the Gram-positive sequences. These two gro ups also miss a signature segment [about 25 amino acids (aa) long] pre sent in all other HSP70 species (Gupta and Golding 1993), We observed a second signature sequence of about 4 aa absent from all eukaryotic h omologues, significantly aligned in all prokaryotic sequences. Consens us sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gramnegative, singular bacteria, mitochondria, plastid s, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplas mic isoforms]. All group consensus comparisons tend to summarize bette r the alignments than do the individual sequence comparisons. The glob al individual consensus ''matches'' 87% with the consensus of consensu ses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carbo xyl terminus of the sequence highlighting the hypercharge run EEDKKRRE R (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contr ast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikin gly, several of the eukaryotic isoforms show multiple mixed charged cl usters. These clusters were interpreted with supporting data related t o HSP70 activity in facilitating chaperone, transport, and secretion f unction. We observed that the consensus contains only a single tryptop han residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, sugge sting a polyphyletic split of Trypanosoma and Leishmania protist mitoc hondrial (Mt) homologues separated from Mt-animal/fungal/plant homolog ues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba h istolytica and Trichomonas vaginalis species were analyzed. The E. his tolytica HSP70 is most similar to the higher eukaryotic cytoplasmic se quences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplas t sequences. This appears to be at variance with the hypothesis that E . histolytica rather recently lost its mitochondrial organelle. T. vag inalis contains two HSP70 sequences, one Mt-like and the second simila r to eukaryotic cytoplasmic sequences suggesting two diverse origins.