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Volume 198, number 1 FEBS 3506
Review-Hypothesis
Why mitochondria need
March 1986
a genome
Gunnar von Heijne
Research Group for Theoreti cal Biophy sics, Department of
Theoretical Ph ysi cs, Roy al Inst it ut e of Technology,S-100 44
Stockholm, Sweden
Received 2 December 1985; revised version received 10 January
1986
The evolution of the mitochondrial genome towards the compact
organization found in the higher eukaryo-tes is discussed. It is
suggested that the machinery for co-translational protein export
across the endoplasmicreticulum membrane sets strict limits on the
kinds of protein-coding genes that can be successfully trans-ferred
from the mitochondrial to the nuclear genome. This hypothesis is in
perfect agreement with the pat-tern of mitochondrially vs nuclearly
encoded mitochondrial proteins found in species such as man,
mouse,
and Xenopus.
Mit ochondria Evolut ion Protein export
1. INTRODUCTION protein export in eukaryotic cells [5],
export
The mitochondrial genome of higher eukaryotesis extremely
economically organized [l-3]. Itmakes use of only 22 tRNAs. It
codes for no morethan 13 proteins, the overwhelming majority
ofmitochondrial proteins being encoded in the cell
nucleus and imported into the mitochondrion in apredominantly
post-translational fashion [4].There are no introns or spacers
between thegenes, and a few genes even overlap. These obser-vations
indicate that the mitochondrial genome inthese organisms has been
stripped of all elementsthat could be either moved to the nucleus
ordispensed with altogether; but why do some genesstill remain?
Here, I show that only one assump-tion is needed to account for the
existence andcomposition of the present-day mitochondrial
genome, namely that the mechanism of post-translational
mitochondrial protein import evolvedonly after the signal
peptide-dependent machineryfor co-translational protein export from
the cellhad come into existence.
2. PROTEIN SECRETION AND MEMBRANEPROTEIN BIOGENESIS
According to the currently accepted model for
through the endoplasmic reticulum is initiated
co-translationally as soon as a hydrophobic leaderpeptide [6] (or
signal sequence) emerges from theribosome. This leader peptide may
subsequently becleaved from the mature chain by a leader pep-tidase
(thus producing either a secreted soluble
protein or an internally anchored membrane pro-tein), or may
resist cleavage to yield an N-terminally anchored protein. In fact,
it has beenshown that an N-terminal transmembrane segmentcan
initiate export and permanently anchor anunrelated protein to the
membrane when fused toits N-terminus [7]. A sufficiently long
internalhydrophobic stretch apparently can also initiateexport,
resulting in a protein with a large cytosolicN-terminal domain
[19,201. Such internal stretcheshave also been shown to initiate
export in the nor-
mal way when artificially moved to the N-terminusof the protein
[21]. A hydrophobic segment no fur-ther than 70-90 residues from
the C-terminus,however, is not expected to activate the
co-translational export machinery, since the wholeprotein will be
finished by the time such a segmentemerges from the ribosome.
Finally, the only dif-ference between an N-terminal, cleavable
signalpeptide and a (N-terminal or internal) permanentanchor
segment found so far is the length of the
Published by Elsevier Science Publishers B. V. (Biomedical
Division)
00145793/86/%3.50 0 1986 Federation of European Biochemical
Societies 1
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March 1986olume 198, number 1 FEBS LETTERS
Table 1
Hydrophobicity analysis of mitochondrially and nuclearly encoded
proteins
Protein No. of Signal N-Cmembrane sequence distancesegments
Protein No. of Signal N-Cmembrane sequence distancesegments
Mitochondrial genes:Xenopus
Cytochrome coxidase I
Cytochrome coxidase II
Cytochrome coxidase III
Cytochrome bATPase 6URFlURF2URF3URF4URF4LURFSURF6URFA6L
YeastATPase 8ATPase 9Hypothetical protein
B-386Ribosomal protein var 1
Cytochrome bmaturase (intron)
oxi intron 1 proteinoxi intron 2 proteinoxi intron 3 proteinoxi
intron 4 proteincob intron 4 protein21 S RNA intron
protein
Nuclear genes:Yeast MSS 51Yeast porinYeast 70 kDa proteinYeast
cytochrome clYeast cytochrome c
oxidase IVYeast cytochrome c
oxidase VYeast cytochrome c
oxidase VIYeast cytochrome c
oxidase VIII
2
8-12 56 499
2- 3
3- 6 20 2256-10 46 3456- 6 9 2136- 9 11 3134- 9 205 1673- 3 1
1094-10 4 4002- 3 1 95
14-15 9 6004- 4 1 170I- 1 6 46
l- 1 13 302- 2 14 60
o- 0o- 0
3- 6 32 389I- 3 14 820o- 3 31 762o- 0 17 _
3- 6 22 455l- 0 258 _
o- 0
o- 0o- 0o- 0l- 0
o- 0
l- 1
o- 0
I- 1
_ _
__
__
__
__
_ _
98 58
_
27+ts
-
20
27
__
202
_-
Yeast manganesesuperoxide dismutase
Yeast cytochrome cperoxidase
Yeast cytochrome creductase, 14 kDa
Yeast cytochrome creductase, 17 kDa
Yeast citrate synthaseYeast cytochrome cYeast EF-TuYeast ilvl
proteinNeurospora ATPase 9Neurospora Rieske
2Fe-2S proteinNicotiana ATPase pRat carbamoyl-
phosphatesynthetase I
Rat ornithinetranscarbamylase
Pig malatedehydrogenase
Pig aspartate
transferasePig citrate synthasePig 3-hydroxyacyl-CoA
dehydrogenaseBovine ATPase P-chainBovine ATPase
inhibitorBovine cytochrome
P450 (SCC)Bovine ATPase 9Bovine coupling
factor 6Bovine cytochrome c
oxidase IVBovine cytochrome c
oxidase VBovine cytochrome c
oxidase VIIBovine uncoupling
proteinBovine ADP/ATP
carrier proteinBovine adrenodoxinBovine rhodanese
o- 0
o- 0
o- 0
o- 0l- 0o- 0o- 0l- 42- 2
o- 0o- 0
o- 0
o- 0
o- 0
o- 0o- 0
o- 0o- 0
o- 0
o- 02- 2
o- 0
l- 1
o- 0
o- 0
2- 4
l- 4o- 0o- 0
_
_
_
_
131_-
39391
68_
_
334
_
378_
__
-
_
58+ts
_
102
_
_
215+ts
281 +ts__
_
-
_
_
(48)_-
(346)59
__
_
_
-
_-
__
_
-
21
-
67
-
_
90
16-_
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Volume 198, number 1 FESS LETTERS March 1986
hydrophobic stretch: it ranges approximately from7 to 15
residues in &he former and from I5 to 25residues in the latter
f&12]. There appear to be nostrong constraints on the sequence
of thehydrophobic stretch as long as the length and
minimum hydrophobicity requirements are met[6], but internal
signal peptide-like segments thatare not long enough to qualify as
transmembranehehces apparently cannot initiate export 113).
These properties of the present-day co-translational protein
export machinery suggest thati f this machinery was already in
existence when thefirst mit~hondria appeared, only those
mitochon-drial genes whose protein products did nof containany
potential hydrophobic export signals couldhave been successfully
moved into the nucleus.
3. ANALYSIS OF MITOCHONDRIALPROTEINS
To test this hypothesis, 24 mitochondrialiy and37 nuclearly
encoded mitochondrial proteins wereextracted from the National
Biomedical ResearchFoundation protein sequence data bank
(version6.0, September 19g5) and searched for the ex-istence of
hydrophobic regions that could serve astargeting signals for export
across the endoplasmicreticulum. As shown in table 1, al l of
the
mitochondrially encoded proteins from Xenopusand most of those
from yeast, but only a few of thenuclearly encoded ones are
predicted to be intrinsicmembrane proteins with long,
hydrophobicmembrane-spanning regions. Moreover, the fewimported
membrane proteins found have theirhydrophobic regions located
within -90 residuesof their C-termini, whereas the mitochondrially
en-coded proteins are predicted to have membrane-spanning segments
all through their sequences.Signal sequence-like segments are also
present near
the N-terminus of most of the mitochondrially en-coded proteins,
but are only found far from it inthe nuclearly encoded group.
Fig. 1. Length vs total hydrophobicity [121 (in kcabmol)of the
most hydrophobic predicted transmembranesegment in the C-terminal
(N-C distance 5 90 residues)and N-terminal regions of each of the
mitochondriallyand nuclearly encoded proteins in table 1.
(x)Mitochondriai C-terminal segments, (+) mitochondrialN-terminal
segments, ft) m&ear C-terminal segments,(0) nuclear N-terminai
segments. Five nuciearfyencoded proteins fl+fgf with suggested
N-terminaltransmembrane segments not so predicted by themethods
employed here have also been included in the
fast group.
These findings are further illustrated in fig. 1,
where the length and total hydrophobicity of themost hydrophobic
C-terminal (N-C distance 6 90residues) and N-terminal (N-C distance
> 90residues) segments have been plotted for each pro-tein in
the mitochondrial and nuclear groups. It isclear that long
hydrophobic segments are foundthroughout the mitochondrial
sequences, but onlyin the C-terminal region of the nuclear
ones.
Thus, noble of the mitochondrially encodedXenopus proteins in
table 1 would be expected tobe able to escape co-translational
routing into the
export pathway were they to be made by cytosolicribosomes, the
only possible exception being theURFAGL protein which probably is
too short to be
The mitochondriahy encoded proteins are from the compIete
Xenopus genome [I] and from yeast (not includingproteins homologous
to Xenopus). ~ydrophobicity anaIysis was carried out according to
Kyte and Doolittle [8] witha span length of 19 residues and the
requirement that {HI) z i A for predicting membrane segments (first
entry), andEisenberg et al. fl If (span Iength = 21, second entry].
Putative signal sequence-like segments were searched For bylooking
for the most N-terminal segment with a fo&ri hydrophob~city h 7
kcaf/mol for span lengths in the interval 7-Hresidues as described
elsewhere [12J (+ ts indicates that the N-terminal mitochondrial
targeting sequence is unknown].The N-C distance is the number of
residues between the first residue of the most N-terminal of the
predicted membranesegments that is also predicted to be a signal
sequence, and the C-terminus {parentheses indicate that the protein
is
probably erroneously predicted to be a transmembrane
protein)
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Volume 198, number 1 FEBS LETTERS March 1986
co-translationally exported. Interestingly, this pro-tein
overlaps the coding region for the ATPasesubunit 6 by lo-46
nucleotides in the Xenopus,mouse, bovine, and human
mitochondrialgenomes [l]; this overlap may effectively lock the
URFA6L gene to the mitochondrion.Although the yeast genome is
less streamlinedthan those of the higher eukaryotes, most of
themitochondrially encoded yeast proteins similarlyseem to be
permanently confined to the mitochon-drial genome by virtue of
their hydrophobicsegments; this is even the case for many of
theintron-encoded proteins (table 1).
4. CONCLUSION
The need for a minimal mitochondrial genomein the higher
eukaryotes and the fact that onlyhighly hydrophobic proteins are
retained in thisgenome can be explained if it is assumed that
anygene successfully transferred from the mitochon-drial to the
nuclear genome during evolution hadto code for a protein that could
escape a pre-existing co-translational export mechanism.
Ex-perimentally, this means that fusing a mitochon-drial targeting
sequence onto a mitochondrially en-coded protein and transferring
the resulting geneto the nucleus would give rise to a protein
that
might be possible to import into mitochondria invitro but not in
vivo (since even markedlyhydrophobic proteins such as the ATPase
subunit9 from Neurospora can be post-translationally im-ported,
there may be no strong sequence con-straints on import once a
protein gets as far as themitochondrial outer membrane).
Conversely, tag-ging a sufficiently long C-terminal extension on
toa nuclearly encoded mitochondrial membrane pro-tein such as
cytochrome c oxidase subunit VIIIwould be predicted to misroute it
into the exportpathway (had mitochondrial targeting sequencesbeen
C-terminal rather than N-terminal, the genefor the coxVII1 protein
would presumably still befound in the mitochondrion).
Finally, if, as seems to be the case, there is muchto be gained
by the cell from reducing the size ofthe mitochondrial genome, the
need for thisgenome only because nuclearly encoded mitochon-drial
proteins have to be compatible with a pre-existing co-translational
export machinery isperhaps the best example yet of a lack of global
op-
4
timization of cellular function at the molecularlevel.
ACKNOWLEDGEMENT
This work was supported by a grant from theSwedish Natural
Sciences Research Council.
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