LETTERS TIBS 2 1 - A U G U S T 1 9 9 6

the kinetics of H202-induced IRP1 activation have incriminated other cellular activities either as 'accomplices' that facilitate the oxidant attack on the cluster or even as the sole 'perpetrators ' : in cultured cells, partial IRP1 activation is evident within 15 min of H202 exposure and complete activation requires treatment for 45-60 min. When H202 is removed after 15 min and cells are chased for an additional 45 min, complete IRP1 activation ensues as if the peroxide had not been removed ~S. This biphasic activation pattern with an H202-triggered 'induction phase' and a subsequent 'execution phase ' would not have been predicted by the 'direct attack' scenario.

I suggest two additional mechanisms by which oxidant stress could induce the removal of the iron-sulfur cluster from IRP1 that accommodate the above experimental findings. One of these models takes the extreme opposite view to the 'direct attack' theory: oxidants play no role in destabilizing the cluster chemically, but induce a cellular activity that will then remove the cluster (Fig. lb). The other model integrates the first two: a cellular factor, activated after H202 exposure of cells, acts alongside oxidants to mount a joint attack on the cluster,

where the factor facilitates the oxidant attack and the two act as accomplices in cluster removal (Fig. lc). Even if the evidence appears to implicate the latter models to explain the activation of IRP 1 by oxidative stress, all three models clearly underscore the suitability of iron- sulfur clusters as regulatory switches for responses to altered oxygen tension or oxidative stress. Moreover, different forms of reactive oxygen intermediates or NO appear to operate iron-sulfur switches in highly specific ways; for example, SoxR is activated by superoxide and NO (but not H202). Even more strikingly, IRP1 is regulated by both NO and H202, but the two control IRP 1 activity by apparently different mechanisms 15. Whether iron-sulfur clusters serve as biosensors (Fig. la), respondents (Fig. lb) or integration points (Fig. lc) might therefore vary from example to example, and surely become a focal topic for future research. The jury is still out on the 'attack on the cluster ' case.

References 1 Martins, E. A., Robalinho, R. L. and Meneghini, R.

(1995) Arch. Biochem. Biophys. 316,128-134 2 Pantopoulos, K. and Hentze, M. W. (1995)

EMBO J. 14, 2917-2924 3 Hentze, M. W. and Argos, P. (1991) Nucleic

Acids Res. 19, 1739-1740 4 Rouault, T. A. et al. (1991) Cell 64, 881-883 5 Kennedy, M. C., Mende-Mueller, L.,

Blondin, G. A. and Beinert, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11730-11734

6 Khoroshilova, N., Beinert, H. and Kiley, P. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2499-2503

7 Lazazzera, B. et at. (1996) J. Biol. Chem. 271, 2762-2768

8 Rouault, 1. A. and Klausner, R. D. (1996) Trends Biochem. Sci. 21, 174-177

9 Hidalgo, E. and Demple, B. (1996) J. Biol. Chem. 271, 7269-7272

10 Hidalgo, E. and Demple, B. (1994) EMBO J. 13, 138-146

11 Drapier, J-C. et al. (1993) EMBO J. 12, 3643-3649

12 Weiss, G. et al. (1993) EMBO J. 12, 3651-3657 13 Pantopoulos, K. and Hentze, M. W. (1995) Proc.

Natl. Acad. Sci. U. S. A. 92, 1267-1271 14 Bouton, C., Raveau, M. and Drapier, J-C. (1996)

J. Biol. Chem. 271, 2300-2306 15 Pantopoulos, K., Weiss, G. and Hentze, M. W.

(1996) Mol. Cell. Biol. 16, 3781-3788

MATTHIAS W. HENTZE

Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Email: hentze@embl-heidelberg.de

Reply We have outlined some general features of iron-sulfur proteins that might allow them to function directly as sensors of oxygen and/or other oxidants. In addition, we described and referenced several examples in which oxidation leads to degradation of the iron-sulfur cluster of a purified protein in vitro. Though greater complexity is always possible in cellular regulatory systems, we find the inherent simplicity of sensing based on oxidation and spontaneous degradation of iron-sulfur clusters attractive.

Kinetic and pharmacological arguments have been advanced (see above) that have been interpreted to support the existence of a signalling pathway, but we believe that these experiments are open to many different interpretations. The time course of any drug response will be influenced by the rate of absorption, distribution within the cell, rate of biotransformation and rate of excretion of the drug. Moreover, most drugs have multiple targets. We have found that many doses of hydrogen peroxide that activate IRE binding in cells also significantly impair protein biosynthesis as assessed by incorporation of radioactive amino acids.

If cells are dying or recovering from an acute insult in the course of pharmacological manipulation, it is very difficult to draw specific conclusions about the treatment. We await biochemical or genetic characterization of intermediates in a putative signaling pathway involved in disassembly of the iron-sulfur cluster of IRP1.

TRACEY ROUAULT AND RICHARD KLAUSNER

Section on Human Iron Metabolism, Cell Biology and Metabolism Branch, NICHHD, Bldg 18, Room 101, Bethesda, MD 20892, USA.

Do non-long terminal repeat retrotransposons have nuclease activity? A high level of homology has been found between regions of the non-long terminal repeat (LTR) retrotransposons (non-site-specific) and the AP endonuclease protein family. Nine highly conserved domains in non-LTR retrotransposons (Fig. 1) were detected. At least 60% of the amino acids that form the consensus motif in AP family

�9 1996, Elsevier Science Ltd

nucleases 1 are maintained in similar relative positions in non-LTR elements. Interestingly, the amino acid Asn153 of exonuclease III, which hydrogen bonds to the nucleotide 03 ' position of the DNA and interacts with the 5'-phosphate group 2, is present in all non-site-specific non-LTR retrotransposons (indicated by �9 in Fig. 1). In addition, the residues Asp229, Ser257 and His259 of exo 111, which are thought to be directly involved in the hydrolytic cleavage of the P-O3' bond, and the metal ion-bound Glu34 are also present in these elements (indicated by * in Fig. 1).

When we compared the consensus sequence of the AP protein family and of the non-LTR elements with sequences present in the SWlSS-PROT database, we detected similarities with particular domains of the DNases I of different organisms 3-6. The Z-score ranges between 3.7 and 7. Figure 1 shows that eight out of the 12 amino acids, described as forming the DNA interaction domain of DNase I (Ref. 7), are conserved. The residue, proposed by Weston e t al. 7 to be involved in DNase I acid-base catalysis (His252), and Asp168 and Asn170, which are in contact with the scissfle phosphate

283

AP family

TIBS 2i - AUGUST 1996

Retro- transposon

DNase I

AP family

Retro- transposon

DNase I

AP family

N74 Y76

Retro- transposon

EX3 ECOLI EXOA BACSU RRP1 DROME APE1 HUMAN

ZMCIN4E G3 DMLINEJA AATRANSA LINI_HUMAN TCRNANLTR NLRICTH R 1BM TART-B1

DRNI_BOVlN

EX3 ECOLI EXOA BACSU RRP1 DROME APE1 HUMAN

ZMCIN4E G3 DMLINEJA AATRANSA LIN1 HUMAN TCRNANLTR N LR 1CTH R 1Bm TART-B1

DRN1 BOVIN

EX3_ECOLI EXOA BACSU RRPI_DROME APEI_HUMAN

ZMCIN4E G3 DMLINEJA AATRANSA LIN1 HUMAN TCRNANLTR NLR1CTH R1Bm TART-B1

DNase I DRN1 BOVIN

domain I

WN g L RA

6 F N I NG L RAIR

6 W N V N G L RAIV 432 WNVAG L RAIW

i0 A R G LIT m

6 L N A R S L N SG 13 LNVNG LN

1 0 6 ~ N V S i LL~'r""~, 13 WNAN

16 I N LG

15 WNAG G I N "1

28 N I R T F~G~

R9 El3

M R K I L K K I I K K I

A K R I T K . I .R~

KEI KRI K

TR

T KI

TI4

(3) (3) (2) (2)

(2) (i) (3) (2) (2) (0)

(2) (i)

(3)

(9)

domain III

G y G v 1

HdGVALL

YSGTAVF YAGVA.I YSGVGLL

IG .SGGILI G S S R G G G V L Si N S R G G S ~ V

CGGVAII ..GVAIL

.GGVAVL

KGGVAII K A G V [ ~ [ ~ RGGVA~I

p vl

L L F V gFIA KFFV HTLI

PLLL NY ~ I RVV I KFIL

DVML

e

13 I (12) (13)

(12)

L V (i0) I I (12)

(ii)

(9) (i4)

( i i )

(i4)

L F~R~ (15)

LETTERS domain II

1 e p D i 1 Q E T K

7 Fldv R R 0 ; o v i IRN ,19, L .IS YL KE E DAD I I C LQ E T K ~ D I (18)

1~ T, I DIXIE g P D I F C L Q s T ~ I ~ V ~ I (22~

v Rbi[-4]~Ds ,[~TDvc L o ~ �9 ~ I~]N[ (23) F H L A, AJV S g A DIFID L Z A L T s T W LVL~ ~ (22) L R IIF L S D H DL~JD V M L T T E T H M RIV (17)

{18) LAISW I K S Q D P S VC C I Q E T H LLMC (21) L AI.[~]L M A Q G A D I I A I Q E T W ~ (19)

L K~IFLqNWD A H I I I V[-T-]E~R-L S (19)

vL IIT s R. [qv 0 V RF]S HI (28,

domain IV

d g R I a E f

E A Q RR I I MA E (7) VT H D Q V I T L E (3) V F

D D M I T A E (3) F Y H D Q V I VA E (3) F V

R DR DIR L E Y I C V R (7) L Y ND R RVH L Q (5) VT KV F . . L GV S (7) Y T

RDK YMMVK (7) L T

L VVQVA (6) L I M I~I KV (7) -~ H

AIPII V L . (7)

c s s {7) s H

Rill

E39 R41 $43

domain V r r

Y P n

V I NGY F P Q G V M TrY T P N S L I NV Y V P N S L V TA Y V P N A

L TAVY G P Q Q

V MVVY I P P Q V AAVYL P PA F I A A Y L P[~Q I LNMYA PNT

V A S AYMR P P I L SVY L P RG M V S A Y F Q["Y']S I A S V Y C P P S

ST E A I

domain VI domain VII �9 �9 �9

GD Nv h iD yt w

LIMGDMNII ~ S T D L ~ (49)

I L C G D L N I V A Q E I D (43)IYSWW I VICGDMNIVS MPID (43)IYTFW 1 V L C G D L NIV A E E I D (43)F ~ W l

L I L G D F N I M I R V G E {32)]FTy__~ IGDFNIQPSISWS {41)IHIIN.

G G D Y NIA K~A W W G (29)IF~S~ I G D F NIA K ~ R S W N (23)1Y P ~ S I MGDFNITPLSTLR (36)IYTF ~

C O D F NIM H ~ P Q W E (31) ~ T Y < CGDFNIA K~ REWN (25) PITY ~ C k D~NIA H S . . . P (41) PIT F_.F..~

HIG D F NIA D C S Y V~ (33) Y~__~R I

domain VIII . D y L S

14) I DILIL L A S QI (21 14)ITDIYFIZ~VVS~I (17 14) IL Ply CrSJT. V S (17 14) L DIYIF L L S (17

I 10)II DIRIL M A T (18

(6) ~, ~,. IL ,IVlA /21 (8) IL DI.IF F I T C (16 23) I D I . I L V L T D~ (15 (9) I DIHII L G S !E (16

(9)II DI. L[LIT-----~S K (14 i0) I DI. I L M L S (16

10)II DI. C (16

(i5) A AIP F (13

domain IX

s D H P i

(31) (28) (28) (28)

(20) (26) (17) (2 i ) (19)

(18)

(22) (20) (23)

(3i)

S D H A P V W A S D H C P V E ~

SDHCPITI

S D H C P I T L

S D H S P L L M S [ ] H P A L D F S D H L P I . L S D H V P V T F

S D H S A I K L S D H Y V L T F S D H C A V Y F SDHRLIIVF S D H L P I L L

S D H Y PV E V

DI68NI70 Y175 Y211 H252

i~ 266 250

677

315

O~ 1292 N I 323 A V 219 Q I 217

E L 237 T L 318 Q I 227 G V 220 E I 237

T L 259

Figure 1 The SWlSS-PROT and GenBank/EMBL databases were searched with FASTA, TFASTA and BLAST 9,1~ BESTFIT and COMPARE from GCG were used to compare the members of the AP endonuclease family with each of the retrotransposon sequences analysed. A two-way search resulted in identical findings. The statistical significance, given as Z-scorel% was determined after comparison Of the sequence under investigation with 100 randomly permuted versions of potentially related sequences. The Z-score values of the homologous domains range between 6 and 12. Homology with DNase I was obtained by establishing a profile between the AP family and the non-long terminal repeat (LTR) retrotrans- posons, using PROFILEMAKE 12. The profile obtained was then used in PROFILESEARCH 13 to search for homologies in the SWISS@ROT database. The alignment of the amino acid sequences of the AP family, non-LTR retrotransposons and DNases I were carried out using PILEUP ~4 from GCG and a manual adjustment when necessary. Only one sequence of each subfamily of nucleases and retrotransposons is shown. Blocks of sequence homology were indicated only when the amino acid is conserved in at least three AP proteins and 14 non-LTR retrotransposons. The amino acids were considered to be conserved when they had positive punctuation in the Dayhoff table 15. For clarity we only show the domains of high homology. Numbers in parentheses represent the number of the amino acid residues between the con- served blocks. At the top of the figure, the consensus sequence for the AP proteins described by Seki et a13 is shown. The amino acids in- volved in the binding between DNase I and the DNA duplex, and those involved in the acid-base catalysis of DNase I (Refs 7, 16) are shown at the bottom of the figure. �9 and * represent the active site residues in exonuclease III (Ref. 2). The residue number of each sequence is shown at the beginning and at the end of the sequence.

284

LETTERS TIBS 21 - AUGUST 1996

group, are fully conserved in all of the non-LTR retrotransposons. Further support for this similarity has been determined from crystallographic analyses, which show that the fold of exo Ill is similar to that of DNase I, despite having less than 20% overall sequence similarity 2.

The presence of the exo 111 active residues and the conserved amino acids involved in DNase I acid-base catalysis, in similar regions of the non-site-specific non-LTR retrotransposons, provides an even stronger argument in favor of the potential nuclease activity of these elements. Within this framework, the potential endonuclease activity could be responsible for generating the 3'-OH sites necessary as primers for its reverse transcription (first s tep in the integration mechanism). This would render the existence of nicks in the DNA necessary for the integration of the non-LTR elements. As the non-LTR retrotransposons share DNase I conserved domains, it is also attractive to think that these elements might recognize sequence-dependent structural variations similar to those recognized by DNase I

(Ref. 8) and generate the 3'-OH needed for transposition.

Acknowledgements We are grateful to H. Ramirez for his

collaboration and technical assistance. F. M. and M. O. were supported by a CICYT and PAI Predoctoral Fellowship, respectively. This work was supported by B[O93-0043 Grant from Plan Nacional [+D (CICYT), Spain.

References 1 Seki, S. et al. (1992) Biochim. Biophys. Acta

1131, 287-299 2 Mol, C. D. et al. (1995) Nature 374,

381-386 3 Liao, T. H., Salnikow, J., Moore, S. and

Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495

4 Paudel, H. K. and Liao, T. H. (1986) J. Biol. Chem. 261, 16006-16011

5 Paudel, H. K. and Liao, T. H. (1986) J. Biol. Chem. 261, 16012-16017

6 Shak, S. et al. (1990) Prec. Natl. Acad. Sci. U.S.A. 87, 9188-9192

7 Weston, S. A., Lahm, A. and Suck, D. (1992) J. Mol. Biol. 226, 1237-1256

8 Lomonossoff, G. P., Butler, R J. G. and KIug, A. (1981) J. Mol, Biol. 149, 745-760

9 Pearson, W. R. and Lipman, D. J. (1988) Prec.

Natl. Acad. Sci. U. S. A. 85, 2444-2448 10 AItschul, S. F. et al. (1990) J. Mot, Biol. 215,

403-410 11 Doolittle, R. R. (1981) Science 214, 149-159 12 Gribskov, M., McLachlan, A. D. and Eisenberg,

D. (1987) Prec. Natl. Acad. Sci. U. S. A. 84(13), 4355-4358

13 Gribskov, M., Luthy, R. and Eisenberg, D. (1990) Methods Enzymol. 183, 146-159

14 Feng, D. F. and Doolittle, R. F. (1987) J. MoI. Evol. 25, 351-360

15 Schwartz, R. M. and Dayhoff, M. O. (1979) in Atlas of Protein Sequence and Structure (Dayhoff, M. 0., ed.), pp. 353-358, National Biomedical Research Foundation

16 Suck, D., Lahm, A. and Oefner, C. (1988) Nature 332,464-468

FRANCISCO MARTIN, M6NICA OLIVARES AND MANUEL C. LOPEZ

Departamento de Biologia Molecular, Institute de ParasitologTa y Biomedicina, CSIC, Ventanilla, n o 11, 18001 Granada, Spain. Email: mlopez@samba.cnb.uam.es

CARLOS ALONSO

Centre de Biologia Molecular, CSlC, Cantoblanco, 28049 Madrid, Spain.

Methionine aminopeptidase-l: the MAP of the mitochondrion? Methionine aminopeptidases (MetAPs or proteins encoded by MAP genes) are ubiquitous enzymes that cleave the amino-terminal methionine from many newly translated polypeptides. In

eubacteria, a single MetAP is generally sufficient to fulfil this role, but eukaryotic genomes contain two distantly related enzymes, which, at least in yeast, are both required for normal growth L2. One of these proteins, MetAP-1, closely resembles the enzymes known from eubacteria, but the other, MetAP-2, is only distantly related to other MetAPs (Refs 1, 2).

Recently, putative MAP genes have been identified from two archaebacteria,

one a fragment from Methanothermus fervidus 3, and the other a full length gene from the distantly related thermophile, Sulfolobus solfataricus (C. W. Sensen et al., unpublished). The predicted protein products of these archaebacterial genes have all the hallmarks of other MetAPs, including the conservation of six residues implicated in the coordination of a cobalt ion co-factor, and the adjacent motifs (Fig. 1). Of all other sequences, the

(a) Eubacterial

Eukaryotic MetAP-1

Archaebacterial

Eukaryotic MetAP-2

DD H E H

II I I I DD H E H ii l l,_l

H E , , - , DD H E H I I I I I

(b) Human-2 ICKI ~ FGTHISGRII

Ratp67 ICKI ~ FGTHISORII Yeast-2 VMKV ~ YGVQVNGNII

Me~o~ermus LVKI D IGVHVDGFIG

Su/fo/obus WKL D LGAHIDGFIS

BacH;us IISI D IGAKLNGYHG M y c o p ~ s m a KLTL ~IIGIDYHGYLC

SynechocysfisA I I N V ~ V T P I V D G Y H G

Synechocyst isC LLKV ~ TGAYFQGYHG

Escherich~ I V N I ~ VTVIKDGFHG Haemophi lus IVNI ~ VTVIKDGYFG

Yeast-1 IVNL ~ VSLYYQGYHA

Human-1 ZVNV ~ ITLYRNGYHG

CAFT

CAFT

SAFT

~ TATT TAIT

~SAWP ~AAFL

CSRF

~SCIA

TSKI NSKI

~LNEY

D LNEF

NLNG i

NLNG I NLCG! NLTG

NLGG

EYV(

EFG(

DFV(

EFT( EYC( EYC(

TYC(

SYC(

SIGQ YAI ~ TFG QFE

H S I G P YAI ~ TFG QFE

~{ S l A P FAI ~ TFG QFE TIDR . . . i . . . . . .

LIRR YAI i~ PFA QFE

GVGQ LAI <~[ PMV HFE GCGI ICI ~ PMV HVE

i~ GISK FTI I PMI QFE

GVGQ LAI ~ PIV QFE GIGR FTI ~ PMV QYE

~{! GVGT FTI ~ PMI QYE

Hi GVGE FTI !~ PMI QFE

H~ GIHK FTI ~ PMI QFE

TIL TIL

TIL

TVI

TIA MYH TIA

TVL

TIV QLI

TLL

TLL

Figure 1 (a) Schematic of methionine aminopepti- dase proteins. The core enzyme is shown in blue, the acidic amino-terminal domain of eukaryotic MetAP-2 is in yellow, the MetAP-1 amino-terminal extension is in red and the 64-amino acid insertion shared between archaebacteria and eu- karyotic MetAP-2 is in green. The identity between the insertions of Sulfolobus and eukaryotes is 58-60%, as compared with a 65.5-70% identity over the rest of the alignable core enzyme (based on 145 pos- itions used in the phylogenetic analysis). Five highly conserved cobalt co-ordinating residues (D, H and E) are also indicated. (b) Amino acid sequences surrounding the five highly conserved cobalt co-ordinating residues (boxed), which appear in the core enzyme at positions 97, 108, 171, 204 and 236, respectively, in the E. coil protein< We include the rat p67 trans- lation factor s in the MetAP-2 class be- cause this enzyme is also likely to be an aminopeptidase: it shares 95% identity with human MetAP-2.

�9 1996, Elsevier Science Ltd Plh S0968-0004(96)20017-9 285