Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes.

Abstract

Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coil, COp. AU members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding. Within these regions there is at least 85% similarity between the most distant members of the family. The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells.Energy-dependent proteolysis plays a key role in prokaryotic and eukaryotic cells by regulating the availability of certain short-lived regulatory proteins, ensuring the proper stoichiometry for multi-protein complexes, and ridding the cell of abnormal proteins (1)(2)(3). A homologous energy-dependent protease shared by evolutionarily diverse organisms has not been previously described. In this paper we provide evidence that the regulatory subunit of the ATP-dependent Clp protease of Escherichia coli has been conserved to an unusual degree in numerous prokaryotes and eukaryotes. The Clp protease of E. coli is a two-component ATP-dependent protease that contributes to the turnover of abnormal proteins (4-7). The large ClpA subunit (81,000 kDa) has intrinsic ATPase activity. The smaller ClpP subunit (21,000 (7,9).Sequencing of the cipA gene, encoding the regulatory subunit of the Cip protease, allowed us to discover that ClpA is a member of a family of well-conserved proteins with no previously described function. Members ofthe family include a second E. coli gene, called clpB, and genes from plants, trypanosomes, and Gram-positive and Gram-negative bacteria. E. coli contains two members of the family (see below)as does tomato (E.P., unpublished data) (Fig. 1). § § T. brucei has one gene with DNA sequence homology to the family (M.C., unpublished data); other organisms listed in Fig. 1 have not been tested for the presence of a second gene. Most of these sequences have not previously been published; the presence in the data banks of a fragment of the C terminus of the clpB gene (previously called ORF-BG) allowed others of us encountering homologous sequences to contact the author of the first publication (11) and subsequently contact each other. In the absence of a concerted effort to detect Clp-like genes in eukaryotic or other prokaryotic organisms, the serendipitous discovery of seven such genes and a fragment of an eighth suggests that the family is extremely widespread. The degree of conservation strongly suggests that the members of this family all represent the regulatory subunit for Clp-like energy-dependent proteases, although the possibility exists that the ClpA protein has evolved other ATPdependent regulatory functions as well.The proteins predicted from the DNA sequences of members of this family have two regions of particularly hig...

Genetic evidence for a functional relationship between Hsp104 and Hsp70

“…The biochemical function of HsplO4 is unknown, but hsp104 mutations do have a specific temperature-related effect on the splicing of mRNA precursors (38) (10,20). ClpA binds to and regulates the activity of the dodecameric ClpP protease in vitro (12,13).…”

Dominant Gain-of-Function Mutations in Hsp104p Reveal Crucial Roles for the Middle Region

“…In fact, it has previously been termed, very naturally, the "spacer" region (Gottesman et al, 1990). It is also highly variable even among members of the B-type HSP100 proteins from plants, bacteria, and fungi (Schirmer et al, 1996), all of which function in stress tolerance (Figure 2).…”

“…The importance of the MR in Hsp104p function was unexpected due to its extreme variability: it is the most diverse region among class 1 HSP100/Clp proteins, in both length and sequence (Gottesman et al, 1990;Schirmer et al, 1996). In fact, it has previously been termed, very naturally, the "spacer" region (Gottesman et al, 1990).…”

“…Unexpectedly, seven of the remaining 12 substitutions clustered in the highly variable MR. In fact, every mutant recovered in the screen for disruption of Hsp104p regulation had at least one MR mutation.Although the amino acid sequence of the MR is highly variable, one small segment of 11 residues (Figure 2) exhibits moderate conservation among those members of the HSP100 family that function in thermotolerance (including plant, bacterial, and fungal members; Gottesman et al, 1990;Schirmer et al, 1996). Five of the seven MR mutations recovered in our screen were located in this small conserved segment (Figures 1, open box under MR; and 2).…”

The 65‐kDa protein derived from the internal translational start site of the clpA gene blocks autodegradation of ClpA by the ATP‐dependent protease Ti in Escherichia coli

“…Accordingly, mutagenesis of the 5'-end AUG codon results in an exclusive synthesis of the 65-kDa protein (referred to as ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produces only the 84-kDa protein. However, ClpA65 lacking the N-terminal 168 amino acids shows little or no ATPase and is unable to support the proteolytic activity of ClpP, although it retains the intact, two highly conserved sequences for ATP-binding like ClpA84 [7].…”