Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4532-4536, May 1995 Cell Biology FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit (protein translocation/quality control/proteolysis/AAA family/membrane protein) AKIO KIHARA, YOSHINORI AKIYAMA, AND KOREAKI ITO Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-01, Japan Communicated by Randy Schekman, University of California, Berkeley, CA, February 13, 1995 (receivedfor review December 5, 1994)
ABSTRACT When secY is overexpressed over secE or secE Overexpression of SecY from a plasmid does not lead to is underexpressed, a fraction of SecY protein is rapidly significant overaccumulation of SecY (2, 12). Under such degraded in vivo. This proteolysis was unaffected in previously conditions, the majority of SecY is degraded with a half-life of described protease-defective mutants examined. We found, about 2 min, whereas the other fraction that corresponds to the however, that some mutations inftsH, encoding a membrane amount seen in the wild-type cell remains stable (2). When protein that belongs to the AAA (ATPase associated with a SecE is co-overproduced, oversynthesized SecY is stabilized variety ofcellular activities) family, stabilized oversynthesized completely (2, 12). Mutational reduction of the quantity of SecY. This stabilization was due to a loss ofFtsH function, and SecE is accompanied by destabilization of the corresponding overproduction of the wild-type FtsH protein accelerated the fraction of newly synthesized SecY molecules (2). These degradation. The ftsH mutations also suppressed, by allevi- observations indicate that uncomplexed SecY is recognized ating proteolysis of an altered form of SecY, the temperature and hydrolyzed by a protease and that SecE can antagonize the sensitivity of the secY24 mutation, which alters SecY such that proteolysis. its interaction with SecE is weakened and it is destabilized at We now report that degradation of SecY requires FtsH, an 42°C. We were able to isolate a number of additional mutants essential membrane protein (13) with its cytoplasmic domain with decreasedftsHexpression orwith an altered form ofFtsH significantly homologous to those of the members of the AAA using selection/screening based on suppression ofsecY24 and (ATPase associated with a variety of cellular activities) family stabilization of oversynthesized SecY. These results indicate recently shown to be widely distributed among eukaryotes and that FtsH is required for degradation of SecY. Overproduction prokaryotes and involved in a variety of cellular functions of SecY in theftsH mutant cells proved to deleteriously affect (13-15). Previously, involvement of FtsH in multiple cellular cell growth and protein export, suggesting that elimination of processes has been suggested, including protein integration uncomplexed SecY is important for optimum protein trans- into membrane (16), protein export (16, 17), and degradation location and for the integrity of the membrane. The primary of A CII protein (18). We discuss what might be the primary role of FtsH is discussed in light of the quite pleiotropic function of FtsH that can lead to the pleiotropic mutational mutational effects, which now include stabilization of uncom- effects, which now include stabilization of SecY. plexed SecY. MATERIALS AND METHODS Elimination of malfolded, misassembled, and other unneces- sary or interfering proteins will help functioning of normal Bacterial Strains. E. coli K-12 strains MC4100 (Alac araD proteins. Such processes, sometimes called "quality control," thiA rpsL relA) and CSH26 (Apro-lac thi) have been described may be especially important for membrane proteins; for in- (19, 20). AD16 was a derivative of CSH26 carryingF'lacIqZM15 stance, subassemblies of potentially channel-forming proteins Y+pro+. AR796 (MC4100,zhd-33::TnlOzhj-3198::TnlOkan) and may lead to uncontrolled collapse of chemical gradients. its isogenic pair, AR797 (MC4100, zhd-33::TnlO zgj-3198:: Prokaryotic cells may provide a suitable system for studying TnlOkan ftsHI), were given to us by T. Ogura (Kumamoto quality control of membrane proteins. Although several nat- University). AK315 (AD16, zgj-231::TnlO ftsHlO1) and urally or conditionally unstable proteins as well as their AK318 (an isogenicftsH+ counterpart of AK315) were trans- responsible proteases have been studied in considerable detail ductants of AD16 that received the zgj-231::TnlO region from in Escherichia coli (1), our knowledge about degradation of AD245 (16). AK345 (MC4100, secY24 zgj-231::TnlOftsHlO1) membrane proteins in this organism is still limited. The present and AK342 (an isogenic ftsH+ counterpart of AK345) were work was aimed at identifying the proteolytic system respon- similarly constructed, using IQ555 (21) as recipient in P1 sible for the rapid degradation of SecY that has failed to transduction. AK421 (MC4100, zhd-33::TnlO secY24 complex with SecE (2) and at studying any phenotypic con- zgj-3198::TnlOkanftsHl) and AK420 (an isogenicftsH+ coun- sequences of accumulation of excess SecY. terpart of AK421) were transductants of IQ85 (22) using SecY and SecE, essential components of protein translocase AR797 as donor and zgj-3198::TnlOkan as a selective marker. of E. coli (see ref. 3 for review), span the cytoplasmic mem- Isolation of Mutants Defective in Degradation of Excess brane 10 times and 3 times, respectively (4, 5). Together with SecY. Cells of AK406 (AD16, secY24 zhd-33::TnlO, con- SecG they form a complex (6, 7). Genetic evidence suggests the structed by P1 transduction) were plated on minimal E agar at importance of cytoplasmic domains of SecY and SecE for their 42°C (21). Temperature-resistant colonies that appeared at interaction (8), while their transmembrane segments may also frequencies of 10-7-10-6 were pooled and transformed with be involved in the interaction (9). pKY258 (secY-lacZa). Transformants were selected at 37°C on SecY and SecE are synthesized roughly in an equimolar ratio peptone agar containing 40 jug of 5-bromo-4-chloro-3-indolyl in wild-type cells (10), and they immediately form a stable P-D-galactopyranoside per ml, 0.25 mM phenylethyl ,3-D- complex that does not measurably dissociate thereafter (2, 11). thiogalactopyranoside, and 1 mM isopropyl P-D-thiogalacto- pyranoside (IPTG), and those with blue colony color (which The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: IPTG, isopropyl ,3-D-thiogalactopyranoside; MBP, accordance with 18 U.S.C. §1734 solely to indicate this fact. maltose-binding protein; Bla, 3-lactamase; IS, insertion sequence. 4532 Downloaded by guest on October 1, 2021 Cell Biology: Kihara et at Proc. Natl. Acad Sci USA 92 (1995) 4533
appeared at frequencies of 5-10%) were saved. Among them, 1 2 3 4 % A 7 Q about 40% showed cold sensitivity for growth, and their re- ,a ;.'~'t ( !i:i,ii...... sponsible mutations (see Fig. 5A) were all found to be linked SecY -ri with zgj-460::Tn5 (see below). They were introduced into AD16 by joint transduction with the transposon. Strains thus Chase (min) 0 0.5 5 20 0 0.5 5 20 constructed were AK646 (AD16, zgj-460::Tn5ftsH102), AK526 (ftsJ100::IS1A), AK521 (ftsJlOl::IS10L), AK520 (zgj-520::ISIA), FIG. 1. Degradation of excess SecY is defective in ftsH mutants. AK524 (zgj-524::IS1A), AK525 (zgj-525::IS1A), and AK523 Cells of AR796 (ftsH+; lanes 1-4) and AR797 (ftsHI; lanes 5-8), each = insertion AK519 was bearing pKY248, were grown at 30°C and shifted to 42°C 1 hr before (zgj-523::ISO1R) (IS sequence). theftsH+ pulse-labeling. Plasmid-encoded secYwas induced for 10 min, and cells counterpart of the above strains. The insertion zgj-460::Tn5 were pulse-labeled with [35S]methionine for 0.5 min followed by chase linked to ftsH (85% cotransduction) was selected from random for the indicated periods. Radioactive SecY was immunoprecipitated, transpositions by joint P1 transduction with the argG+ marker to SDS/PAGE, and visualized. (23). subjected Media. Media used have been described (2, 16, 21, 22). in the ftsH cells; these differences were probably due to the Plasmids. pKY248 and pKY258 were derived from pA- rapid degradation of newly synthesized SecY under the ftsH+ CYC184 and carried secY and secY-lacZa, respectively, under conditions rather than to increased synthesis rates in the the control of the lac promoter (2, 8). pKY318 was similar to mutant cells (2). pKY248, but derived from pBR322 (2). pSTD401 carriedftsH+ Normal FtsH Function Is Required for Degradation of cloned under the lac promoter on pHSG575 (16). pKH58 Excess SecY. When a plasmid carryingftsH+ (pSTD401) was carried theftsH102 allele offtsH; a 0.7-kbApa I-Cla I fragment introduced into the ftsHlO1 mutant or the ftsH+ cells, degra- of PCR products from the ftsH102 mutant chromosome (see dation of excess SecY was accelerated. Fig. 2 compares pulse- below) was used to replace the corresponding fragment of chase profiles of SecY that had been immunoprecipitated pSTD401. using a fixed total radioactivity. The initial incorporation Nucleotide Sequence Determination oftheftsH Region. The should reflect the balance between the rate of translation ftsH coding region was amplified from the chromosome by initiation, chain completion, and degradation. As already PCR, using upstream primer (5'-GAATTCCACAGTTGTA- noted, the rapid proteolysis of SecY results in reduced net ATAAGAGG-3') and downstream primer (5'-GCTCTA- incorporation of radioactivity into SecY (2). In the ftsHlOl GATACAGTCATCTGATGCGG-3'), with attached recogni- mutant, the half-life of excess SecY, estimated from the tion sequences of EcoRI and Xba I, respectively. Amplified was about 12 min SecY was fragments were digested with these enzymes and cloned into degradation phase, (filled circles). pTWV229 (24) for single strand preparation and sequencing. strikingly destabilized in the presence of pSTD401 (open Any deviations from the wild-type sequence were confirmed circles); its half-life was now estimated to be about 1 min by by sequencing at least two independent clones. For amplifi- assuming that the synthesis rate itself equals that obtained by cation of the upstream region of ftsH, the following primers extrapolating the degradation phases of the upper two curves were designed: 5'-CGGACTCTTCTCGTGCAC-3' and 5'- of Fig. 2. Even in the ftsH+ cells, increased FtsH content TCGCCAGCAGGTTTTTACCGGT-3'. The amplified frag- accelerated the degradation of excess SecY, shortening its ments were treated with T4 DNA polymerase and Kpn I and estimated initial half-life from about 2.5 min (filled squares) to ligated with Kpn I-HincII-digested pTWV228 (24). immeasurable (open squares). Pulse-Chase, Immunoprecipitation, and Immunoblotting. These results show that the stabilization of SecY in the Pulse-chase examinations of SecY were done as described (2), ftsHlOl mutant is due to a loss of FtsH function rather than to except that the medium contained glucose; 5 mM cAMP was active interference of the mutant form of FtsH with the included for induction of the lac promoter. Antisera used were a mixture of those the N-terminal and C-terminal 240 _ I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~·~ against ln peptides of SecY, prepared and provided by T. Yoshihisa (this c- A laboratory). Immunoprecipitation of FtsH was similarly con- 200 ducted using anti-FtsH (provided by W. Wickner, Dartmouth Medical School). Immunoblotting of SecY was done using n 160 fixed amounts (1 tg and 3 ag) of total cell proteins essentially .t as described (2). Protein export was examined by pulse-chase 120 labeling, immunoprecipitation of maltose-binding protein o (MBP), OmpA, or 3-lactamase (Bla), and SDS/PAGE as 80n I I I described (25). CU 40 - RESULTS u I I Overproduced SecY Is Stabilized inftsH Mutants. To screen 0 4 8 12 16 mutants defective in the rapid degradation of oversynthesized Chase time (min) SecY, we initially introduced a secY plasmid, pKY248, into several mutants (lon, clpA, clpP, hflA, hflB, spp, ptr, and degP) FIG. 2. Overproduction of FtsH accelerates degradation of SecY. affected in proteolysis. SecY was stabilized in none of them. Cells of AK315 (ftsH101)/pKY318 (plac-secY)/pSTD401 (plac-ftsH) Although the above tests included the hflB29 allele, defective (0), AK315/pKY318/pHSG575 (vector) (0), AK318 (ftsH+)/ in the degradation of A CII protein (18), offtsH, we found that pKY318/pSTD401 (o), and AK318/pKY318/pHSG575 (s) were another ftsH allele, ftsHlOl, stabilized SecY. This mutation grown at 30°C and then induced. Cells were pulse-labeled with was originally isolated as causing a "Std" (stop transfer de- [35S]methionine for 0.5 min and chased for the indicated periods, fective) phenotype to a SecY-PhoA fusion protein (16). Sta- followed by immunoprecipitation of SecY and SDS/PAGE. Radio- of SecY in the mutant was also studied. In activities associated with SecY were quantitated. About 6.5 x 105 cpm bility ftsHl (Ts) of total cell proteins were used in each sample. The symbol (x) on the contrast to excess SecY molecules that were pulse-labeled in ordinate indicates a hypothetical initial labeling, assuming that all the wild-type cells (Fig. 1, lanes 1-4), those labeled in theftsHI labeled chains had been completed and no degradation had taken mutant at 42°C remained intact after chase (Fig. 1, lanes 5-8). place during translation, and was obtained by extrapolating the An apparent increase in the initial labeling of SecY was noticed logarithmically plotted versions of curves 0 and *. Downloaded by guest on October 1, 2021 4534 Cell Biology: Kihara et aL Proc. Natl Acad. Sci. USA 92 (1995)
degradation. The accelerated SecY degradation in the FtsH- A 1 2 3 4 5 6 overproducing cell suggests that FtsH is rate-limiting in deg- radation of excess SecY. This is consistent with the results SecY24 - obtained with the insertion mutations the ex- reducing ftsH B pression and stabilizing SecY (see below). - Mutations inftsH Suppress the Temperature-Sensitive Ex- SecY24 port Defect of the secY24 Mutant by Stabilizing the Mutant Chase (min) 0 1 3 9 27 81 Protein. The secY24 mutation, a Gly-240 -> Asp alteration in cytoplasmic domain 4 of SecY (22), weakens SecY-SecE inter- FIG. 4. The SecY24 mutant protein (chromosomal level) is stabi- action and causes a defect lized by the ftsHI mutation. Cells of AK420 (secY24; A) and AK421 temperature-sensitive export (8). (secY24ftsHl; B) were grown at 30°C and then shifted to 42°C. After The chromosomally encoded SecY+ (without overproduction) 1 hr, cells were pulse-labeled with [35S]methionine for 0.5 min and was stable at 30°C and 42°C (8), but the secY24 mutant protein chased with unlabeled methionine for the indicated periods. SecY was was degraded with a half-life of about 20 min upon exposure immunoprecipitated and separated by SDS/PAGE. to 42°C (ref. 8; see Fig. 4A). When the ftsHlOl mutation was combined with the secY24 dominant character. It complemented partially the tempera- mutation, the resulting strain became able to grow at 42°C on ture sensitivity of the ftsHI mutant at 42°C but not the other minimal E agar, indicating thatftsHlOl suppressed secY24 with cold-sensitive mutants isolated here (see below). Thus, the respect to the temperature sensitivity. Pulse-chase experi- altered FtsH102 protein appears to retain partial activity at ments (Fig. 3) showed that the defects in export of MBP and high but not at low temperature. It was shown that ATPase OmpA were suppressed as well (Fig. 3, compare lanes 9-12 activity of the FtsH102 protein was markedly lower than that with lanes 5-8). of the wild-type FtsH protein (Y.A., unpublished results). The abundance of SecY before and after the temperature The rest of the mutants proved to have insertions of IS shift was examined by immunoblotting. SecY abundance in the elements in the promoter-distal region offtsJ or in the inter- wild-type cells was not affected by the temperatures examined. genic region between ftsJ and ftsH (Fig. 5A). It was reported SecY in the secY24 mutant cells was about 70% and 23% of the thatftsJ andftsH form an operon (28). Pulse-label experiments wild-type level, at 30°C and upon a 1-hr exposure to 42°C, showed that these insertion mutations lowered the expression respectively. The secY24 ftsHlOl double mutant contained levels of at 20°C about 90% and 60% of the wild-type value at 30°C and at 42°C, ftsH, especially (Fig. 5B). respectively. Thus, SecY24 is significantly stabilized in the These results indicate thatftsH is a main factor necessary for double mutant. We also combined the secY24 and the ftsHI degradation of SecY. However, among non-cold-sensitive mutations. Pulse-chase experiments clearly showed that the mutants, we also identified two additional loci that can be chromosomally encoded SecY24 protein was stabilized by the mutated to be SecY-degradation defective (unpublished re- ftsHl mutation at 42°C (Fig. 4B). sults). A Major Class of SecY-Stabilizing Mutations Affects ftsH. ftsH Mutants Are Sensitized to Overproduction ofSecY. The We isolated mutants defective in degradation of SecY24 as results presented above indicate that excess and uncomplexed well as of SecY. We first selected oversynthesized wild-type A P spontaneous revertants, capable of growing at 42°C, from the ftsH102 secY24 mutant. Those revertants in which the temperature (HS) sensitivity was suppressed by stabilization of SecY24 were 'ffiHzzftsHlO z identified by examining j3-galactosidase activity of the SecY- 100 LacZa fusion protein (Materials and Methods). Model exper- 101-- 52 iments showed that of the LacZa was dictated 520,524 stability portion B by the stability of the SecY part of the fusion protein (unpub- 7 lished results). Many of the mutants thus obtained exhibited 300. WT T T ~(9 cold-sensitive growth. All seven independently isolated cold- '- 'SI' IN' SS" 'S 1 6.1 I.U- sensitive mutants were complemented by pSTD401 (ftsH+) 0 200. and mapped in the ftsH region of the chromosome. These Co mutations conferred cold sensitivity when they were trans- Nl- duced into the secY+ background as well. They indeed stabi- .03 100.