Developmental Cell
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Krebs Cycle Moonlights in Caspase Regulation
Adi Minis1 and Hermann Steller1,*
1Strang Laboratory of Apoptosis and Cancer Biology, Howard Hughes Medical Institute, The Rockefeller University, New York,
NY 10065, USA
*Correspondence: steller@mail.rockefeller.edu
http://dx.doi.org/10.1016/j.devcel.2016.03.016
In this issue of Developmental Cell, Aram et al. (2016) identify a mechanism that uses a Krebs cycle protein to
control local activation of a ubiquitin ligase complex at the mitochondrial outer membrane for temporally and
spatially restricted caspase activation during Drosophila sperm differentiation.
Caspases, a conserved family of cysteine
proteases, have been intensely studied as
key executioners of apoptosis, the most
common form of natural cell death. In
addition, caspases also play important
non-apoptotic roles in development and
differentiation (Yi and Yuan, 2009). A strik-
ing example is the non-lethal use of
apoptotic effector caspases for cellular
remodeling (Fuchs and Steller, 2011;
Schuldiner and Yaron, 2015). Apoptotic
proteins are required for the regulated
destruction of axons and dendrites to
sculpt the nervous system, the degrading
of the nucleus and membrane-bound or-
ganelles during lens fiber differentiation,
and terminal differentiation of spermatids.
This raises the intriguing question of how
the activation of apoptotic caspases can
be restricted in time and space to avoid
death of the entire cell. In this issue of
Developmental Cell, Aram et al. (2016)
report a major advance that sheds new
light onto this question. Using Drosophila
melanogaster spermatogenesis as a
model system, the authors uncover an un-
expected role for a Krebs cycle protein,
Succinyl-CoA synthetase b (A-Sb), in pro-
moting temporally and spatially restricted
caspase activation at the mitochondrial
outer membrane (MOM) (Figure 1).
During terminal sperm differentiation in
Drosophila, the vast majority of proteins
are degraded to generate highly special-
ized and minimalistic cells devoid of major
organelles. This process occurs in cysts
containing 64 haploid cells that remain
interconnected due to incomplete cytoki-
nesis (Fuller, 1993). The terminal differen-
tiation of spermatids involves dramatic
cell elongation and separation into indi-
vidual spermatids; hence, this process is
termed ‘‘individualization.’’ Importantly,
this process involves the degradation of
the majority of proteins and organelles
and depends on apoptotic proteins,
including caspase-3-type effector cas-
pases (Arama et al., 2003). Earlier studies
had already revealed the critical impor-
tance of the caspase inhibitor dBruce, the
apoptosome activator Cytochrome C,
and a Cullin-3-based ubiquitin ligase com-
plex (CRL3) for regulating caspase activity
in a non-lethal manner (Arama et al.,
2007). Furthermore, the Arama lab previ-
ously identified a pseudosubstrate inhibi-
tor for CRL3, termed Soti, which inhibits
the activity of this complex in a spatially
graded manner and thereby restricts cas-
pase activity in time and space (Kaplan
et al., 2010). This fine-tuning of caspase
levels is crucial for male fertility, as either
too low or too high caspase activity has
disastrous consequences.
In the current study, Aram et al. (2016)
used a yeast two-hybrid approach fol-
lowed by co-immunoprecipitation experi-
ments to identify an ATP-specific form of
the Succinyl-CoA synthetase b subunit
(A-Sb) as another key regulator of CRL3
activity. Typically, A-Sb is found in the
mitochondrial matrix and functions within
the Krebs cycle. However, in Drosophila
spermatids, a significant portion of A-Sb
is localized on the MOM where it can
bind to CRL3. In a series of elegant exper-
iments that combine cell fractionation, im-
muno-EM, biochemical analyses, domain
swapping experiments, and genetic and
structure-function studies, Aram et al.
(2016) now demonstrate that A-Sb pro-
motes caspase activation by antago-
nizing Soti to activate the CRL3 complex
at the MOM. They also show that the
localization and function of A-Sb in devel-
oping sperm depends on its N-terminal
domain. This is interesting because
although the CRL3-binding domain of
A-Sb, located at its C terminus, is suffi-
cient for caspase activation in S2 culture
Developmental Cell 37, April 4, 2016 ª2016 Elsevier Inc. 1
cells, it is not sufficient for caspase activa-
tion in spermatids. Furthermore, although
the authors suggest that the binding of
A-Sb to the CRL3 complex is important
for its activation by neddylation, their
RNAi experiments show a more promi-
nent effect on the localization to the
mitochondria than on the levels of neddy-
lation. Collectively, these results imply
that Cul3-neddylation and caspase acti-
vation are uncoupled in spermatids and
that another layer of regulation operates
at the MOM. This could be mediated
through an interaction with other mito-
chondrial proteins or lipids, post-transla-
tional modification, or the proximity to
other apoptosis-related proteins that are
necessary for caspase activation, such
as Cytochrome C.
Irrespective of the exact underlying
biochemical mechanism, the current
work by Aram et al. (2016) reveals an
unexpected ‘‘moonlighting’’ function of
A-Sb for the spatio-temporal regulation
of a Cullin-3-based ubiquitin ligase com-
plex and caspase activity. At the same
time, these findings raise many new
and intriguing questions. Foremost, why
would a Krebs cycle protein involve a
function at the surface of mitochondria
to regulate protein degradation? The
important role of mitochondria in the regu-
lation of apoptosis has long been recog-
nized, as a source of regulatory proteins,
but also as a platform for the sequestra-
tion of apoptotic proteins (Fuchs and
Steller, 2011). Furthermore, sperm differ-
entiation in insects involves very dramatic
changes in the structure of mitochondria.
Therefore, it is possible that A-Sb helps
to sense the appropriate metabolic state
for the initiation of caspase activity.
Intriguingly, two additional non-canonical
functions of A-Sb outside mitochondria
have also been suggested, namely in
 Developmental Cell

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plexes. In addition, although the CRL3

complex is clearly important for caspase
activation during sperm differentiation,
there may be other substrates with rele-
vant biological functions. Another impor-
tant unresolved question is how exactly
A-Sb overcomes the inhibition of CRL3
by Soti. Furthermore, it will be interesting
to investigate the precise mechanism
that determines when and how A-Sb be-
comes located to the MOM, since this
appears to be the rate-limiting step in acti-
vating CRL3 in this system. Finally, it will
be important to explore whether these
findings are restricted to Drosophila
sperm differentiation or represent a more
general regulatory mechanism. Given
the tremendous importance of under-
standing both mechanisms that restrict
caspase activity and the regulation of
CRL3 complexes, it is likely that the
answers to many of these questions will
emerge soon.

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H. (2007). PLoS Biol. 5, e251.

Fuchs, Y., and Steller, H. (2011). Cell 147, 742–758.

Figure 1. Spatially Restricted Caspase Activation at the Mitochondrial Membrane during
Sperm Differentiation Fuller, M.T. (1993). In The Development of
(A) The non-lethal activity apoptotic effector caspases is regulated by multiple pathways at the mito- Drosophila melanogaster, M. Bate and A.M. Arias,
chondrial surface. First, Cytochrome C is necessary to promote caspase activation. Second, the pseu- eds. (Cold Spring Harbor Laboratory Press),
dosubstrate Soti and A-Sbt, a testis-specific isoform of the Krebs cycle enzyme Succinyl-CoA synthase b, pp. 71–147.
have antagonistic effects on the activity of a Culin-3-based ubiquitin ligase (CRL3) complex. Activation of
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2 Developmental Cell 37, April 4, 2016