ATP-dependent proteases that also chaperone prote n b ogenesi Carolyn K. Suzuki, Martijn Rep, Jan Maarten van Dijl, Kitaru Suda, Leslie A. Grivell and Gottfded Schatz The ATP-dependent proteases Cip and FtsH from bacteria, as well as mito- chondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteo{ysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may func- tion in the quality control of protein biogenesis. PROTEP~ BREAKDOWN in liver slices wa~ ~ho~:' over 40 years ago, to be par- tially blocked by metabolic inhibitors yet it took a further 20 years before Goldberg and colleagues purified ATP- dependent proteolytic activities from mammalian and bacterial cellsL in the past two decades, the different pathways of energy-dependent proteolysis have been characterized In great detail, but the requirement for ATP has remained less clear. Recent findings suggest that the ATP-dependeuce of proteases con. served between bacteria and mitochon- drla reflects a chaperone-like function, which can act independently of proteoly- sis, We shall first present a general pro- file of these conserved proteases and then a description of their chaperone- like roles. 6enml ¢lm~'tedotlcs of ATP.depen~nt ~ conserved between b ~ e d a and~ Bacteria and mitochondria share three families of ATP-dependent proteases whose members were all first discovered in Escherichia co~~ (Table i; for reviews, ¢. K. Suzuld, K. ~ d e and G. Schatz are at the Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, SwiLTedand;M. Rap is at the Laboratoryfor Molecular Cell Biology,Catholic Universityof Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee,Belgium; 1. M. vm DlJlis at the Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 Haren, The Netherlands; and L A, (lldvell is at the Section for Molecular Biology, University of Amsterdam, Kruislaan318, 1.098 SM Amsterdam, The Netherlands. see Refs 1-3); (1) protease La, also re- ferred to as Lon, the product of the E. coil Ion gene; (2) the two-component Clp (or Ti) protease composed of an in- variable proteolytic component (ClpP) 1,2 and one of two ATPase components (ClpA or ClpX)l.2.4.5; and (3) FtsH, also referred to as HflB 6,7. Lon and CIp are soluble proteins, whereas FtsH is an- chored in the cytoplasmic membrane, spanning it twice. Lon, Cip and FtsH mediate the degradation of a variety of regulatory proteins that control the heat-shock response, the SOS response to DNA damage, capsular polysacchadde synthesis, the lytic-lysogenic cycle of bacteriophage h (Ref. 8) and pro- grammed bacterial cell death ~. L0n/~ ~ia. The E. co//Ion gene encodes a non-essential 88 kDa protein that func- tions as a homo-oligomer of at least four subunits ~.2,~2. The Lon protein is a serine protease with the catalytic serine resi- due located in the carboxy-terminal half of the protein; mutating Ser679 abolishes proteolysis. Lon functions as an endo- protease, cleaving substrates into pep- tides of 5--20ami~o acids, generating few, if any, free residues. The amino-terminal half of Lon contains Box A and B motifs typical of adenine nucleotide-binding sites. The intrinsic ATPase activity of Lon is stimulated by protein substrates and is unaffected by mutational inacti- vation of the proteolytic site. Thus, a protein need not be degraded by Lon to stimulate its ATPase activity. The degra- dation of casein, bovine serum albumin, XN protein and peptides by the isolated Copyright © 1997, Elsevier Science Ltd. All rights reserved. TIBS 22 - APRIL 1997 Lon protease is stimulated by ATP~,~,~2. Recent work has shown that protein degradation by Lon does not require ATP-hydrolysis if the substrate lacks a stable secondary structure ~1. The in vivo targets of Lon include SulA, which is induced by the SOS re- sponse; RcsA, a transcriptional activator of capsular polysaccharide genes; the transposase encoded by the Tn903 trans- poson; the ~,N proteinS; and CodA, a negative regulator of the CcdB killer protein, which facilitates programmed bacterial cell death ~°. The control of regulatory processes by Lon-mediated degradation is clearly illustrated by the consequence of altered SulA turnover. In E. coli, SulA is a nega- tive regulator of septation and has a half-life in wild-type cells of about 1 rain. When its synthesis is induced by DNA damage in Lon-deficient cells, SulA accumulates to levels that inhibit the essential cell-division protein FtsZ from forming annular rings, which are re- quired for proper septum formation. Lon- deficient cells thus form long, inviable filaments, which are a hallmark of the Ion phenotype. In addition to regulatory proteins, Lon also degrades abnormal proteins such as mutant forms of [3- galactosidase or truncated polypeptides formed in puromycin-treated cells ~.2. [ukary0tes. Goldberg and colleagues first identified a Lon-like ATP-dependent proteolytic activity within the matrix of rat liver mttochondriaL Maurizi and col- leagues cloned the gene for the human Lon protease 13, and showed that it en- codes a 106kDa mitochondrial protein with 58% similarity to E. co~~ Lon. Hu- man Lon mRNA was detected in all tis- sues examined. in the yeast Saccharomyces cerevisiae, Lon is essential for normal mitochon- drial function. The yeast LON gone (also referred to as PIMI) 14.Is encodes a 120 kDa protein that exhibits 60% sequence simi- larity to its E. co/i counterpart; the holo- enzyme has been reported to be a homo-hexamer :6. Purified yeast Lon cata- lyses the ATP-dependent degradation of casein ~*'~6, while in isolated mitochon- dria Lon degrades several heterologous proteins targeted to the matrix ~7. In vivo, Lon mediates the degradation of the ~, and ~/subunits of the F~ATPase, the [~ subunit of the matrix processing pepti- dase ~4.~s,~9 as well as several mitochon- drial ribosomal proteins (S. A. Leonhardt and T. L. Mason, unpublished). Deletion of ,~he LON gone renders cells respiration- de!~.cient, inactivates the mitochondrial genetic system, abolishes ATP<lependent 0968-0004/97/$17.00 Pll: S0968-0004(97)01020-7