Download erp013_60_3_combined 709..714 - Journal of Experimental Botany

712 | Commentary
COMMENTARY
Known knowns, known unknowns,
unknown unknowns and the
propagation of scientific enquiry
David C. Logan*
Department of Biology, WP Thompson Building, 112 Science
Place, University of Saskatchewan, Saskatoon SK S7N 5E2,
Canada
Journal of Experimental Botany, Vol. 60, No. 3, pp. 712–714, 2009
doi:10.1093/jxb/erp043
In February 2002, Donald Rumsfeld, the then US Secretary of State for Defence, stated at a Defence Department
briefing: ‘There are known knowns. There are things we
know that we know. There are known unknowns. That is
to say, there are things that we now know we don’t know.
But there are also unknown unknowns. There are things
we do not know we don’t know.’ As a result, he was
almost universally lampooned since many people initially
thought the statement was nonsense. However, careful
examination of the statement reveals that it does make
sense, indeed the concept of the unknown unknown
existed long before Donald Rumsfeld gave it a new
audience.
Much scientific research is based on investigating known
unknowns. In other words, scientists develop a hypothesis
to be tested, and then in an ideal situation experiments are
best designed to test the null hypothesis. At the outset the
researcher does not know whether or not the results will
support the null hypothesis. However, it is common for the
researcher to believe that the result that will be obtained
will be within a range of known possibilities. Occasionally,
however, the result is completely unexpected—it was an
unknown unknown.
There are many known knowns of intracellular protein
targeting and, as with many fields of research, it seems that
the number of known unknowns increase in parallel. The
key determinants for targeting to mitochondria have been
determined, and there are at least four subclasses of
mitochondrial-targeted proteins containing different targeting signals that are directed to different sites within the
mitochondrion (outer membrane, inter-membrane space,
inner membrane, and matrix) by different mechanisms
(Bolender et al., 2008; Whelan and Glaser, 2007) (Fig. 1).
However, much remains unknown, especially in plants. For
example, there are now a number of known unknowns
resulting from a previously unknown unknown: the existence of proteins dually targeted to both plastids and
mitochondria (Peeters and Small, 2001; Ma and Taylor,
2002; Whelan and Glaser, 2007). We now know that dual
targeting to plastids and mitochondria occurs as a result of
ambiguous signal sequences, but we do not know how these
signals are recognized by both organelles, when other
proteins are only recognized by one (Whelan and Glaser,
2007).
The paper by Chatre et al. (2009) in this issue is an
excellent example of research uncovering unknown
unknowns. Typically, investigations into the mechanics of
intracellular protein targeting have been performed using
protein biochemistry, but the investigation by Chatre et al.
(2009) is not typical. If the study had simply been
investigating mitochondrial targeting using in vitro translation of various proteins with altered targeting signals,
detected by protein electrophoresis and immunoblotting,
the results would have been a combination of ‘yes, the
construct targets to mitochondria’ and ‘no, the construct
does not target to mitochondria’. However, because a cell
biological approach was taken, so much more information
was garnered; that is the power of using fluorescent protein
fusions in vivo.
By means of a bioinformatics screen, Chatre et al. (2009)
identified nucleus-encoded proteins that were predicted,
based on the presence of a coiled-coil domain, to be
targeted to the secretory pathway. However, one of the
proteins identified, and named MITS1, was targeted to
mitochondria when a full-length protein fusion was made to
the N-terminus of YFP (Fig. 2A). MITS1, a putative actinbinding protein, was also correctly predicted, by various
* E-mail: [email protected]
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
Commentary | 713
Fig. 1. The plant mitochondrial import machinery. Mitochondrial precursor proteins synthesized in the cytosol are specifically
recognized by receptors on the TOM complex and translocated through the general import pore. Proteins with N-teminal
targeting signals are recognized by receptors in the TIM23 complex, and translocated into the matrix. Oxa further sorts a small
number of proteins imported into the matrix to the inner membrane. Carrier proteins destined for insertion into the inner membrane
interact with Tim9–Tim10 chaperone complexes in the intermembrane space, ferrying it from the TOM complex to the TIM22
complex, where the protein is inserted into the membrane. Outer membrane b-barrel proteins are imported through the TOM
complex and inserted into the outer membrane by SAM. Import components are coloured to indicate their putative
evolutionary origin: ‘eubacterial origin’ denotes those components for which a likely ancestor in the endosymbiont has been
proposed, while ‘eukaryotic origin’ denotes those components with no relevant similarity to bacterial proteins and which might
have developed specifically in the host cell genome during or after the conversion of the endosymbiont to an organelle.
Abbreviations: TOM, translocase of the outer membrane; TIM, translocase of the inner membrane; PAM, presequence associated
motor; SAM, sorting and assembly machinery. Reproduced from Whelan and Glaser (2007) with the kind permission of Blackwell
Publishing.
bioinformatic tools, to be localized to mitochondria.
Further experimentation demonstrated that the predicted
mitochondrial targeting peptide (mTP), residues 1–39, was
sufficient to target YFP to mitochondria (Fig. 2B). In
addition, when only the first 11 amino acids of MITS1
(MITS11–11) was fused to YFP, the fusion protein was
cytosolic, the same result occurred using the first 31 amino
acids (MITS11–31), consistent with the requirement for
a positively charged region in mTPs; in MITS1 this is
towards the C-terminus of the mTP and is preceded by
a hydrophobic region (Fig. 2A, B). So far, so predictable.
However, the research entered the world of the unknown
unknowns when the team started an in-depth exploration of
the targeting determinants of the N-terminal region. When
MITS112–39 was fused to YFP, targeting was to both the
ER and mitochondria demonstrating that residues 1–11,
although not sufficient for targeting to mitochondria, can
overcome targeting to the ER. Dual targeting to mitochondria and the ER is a known known in mammalian cells
(Huang et al., 1999; Ma and Taylor, 2002), and occurs by
means of N-terminal splice variants. It is not known if wildtype MITS1 is dual targeted. In any case, a major difference
between MITS1 and the dual targeted cAMP-dependent
protein kinase anchoring protein, D-AKAP1, in mammals is
that it is the shorter forms of D-AKAP1, those missing an
extreme N-terminal region, that target to mitochondria
(Huang et al., 1999).
The change in MITS1 targeting resulting from changes in
the N-terminal mTP is interesting, but what I think is much
more interesting is the finding that a single distal tryptophan residue (W361) affects targeting. Full-length MITS1
fused to YFP is found to be targeted solely to mitochondria,
but a single W361A mutation (MITS1W361A) redirects the
protein to the ER and Golgi (Fig. 2C). The W361A
mutation also redirects MITS112–573 from the ER and
mitochondria to the cytosol (Fig. 2C). The obvious
questions that arise include: How does this tryptophan
residue affect targeting? How does this one mutation lead
714 | Commentary
Once again, the discovery of a previously unknown unknown
shows us how little we know and leads to the propagation
of a family of known unknowns which can then be tackled
by traditional hypothesis forming and testing, occasionally
throwing-up another unknown unknown, and so the cycle
continues.
References
Bolender N, Sickmann A, Wagner R, Meisinger C, Pfanner N.
2008. Multiple pathways for sorting mitochondrial precursor proteins.
EMBO Reports 9, 42–49.
Chatre L, Matheson LA, Jack AS, Hanton SL, Brandizzi F. 2009.
Efficient mitochondrial targeting relies on co-operation of multiple
protein signals in plants. Journal of Experimental Botany 60,
741–749.
Fig. 2. MITS1-YFP constructs used by Chatre et al. (2009). (A)
Schematic representation of MITS1 and its N-terminal region. (B)
Schematic representations of the constructs used to dissect the
roles of subdomains of the mTP in MITS1. (C) Schematic representations of the constructs used to investigate the role of distal
tryptophan residues. All figures redrawn from Chatre et al. (2009).
to redistribution of a mitochondrial-targeted protein to the
ER and Golgi? Does the mutation lead to increased
interaction of the protein with ER signal recognition
particles (SRPs)? Is there competition for MITS1W361A
between the SRPs and the mitochondrial import machinery?
Huang LJ, Wang L, Ma Y, Durick K, Perkins G, Deerinck TJ,
Ellisman MH, Taylor SS. 1999. NH2-terminal targeting motifs direct
dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either
mitochondria or endoplasmic reticulum. Journal of Cell Biology 145,
951–959.
Ma Y, Taylor S. 2002. A 15-residue bifunctional element in
is required for both endoplasmic reticulum and mitochondrial targeting. Journal of Biological Chemistry 277, 27328–
27336.
D-AKAP1
Peeters N, Small I. 2001. Dual targeting to mitochondria and
chloroplasts. Biochimica et Biophysica Acta 1541, 54–63.
Whelan J, Glaser E. 2007. Import of nuclear-encoded mitochondrial
proteins. In: Logan DC, ed. Plant mitochondria. Oxford: Blackwell
Publishing, 97–140.