and fig. S6), which might represent cytosolic
In cells depleted of SRP for 1 hour, mitochon-
dria lost their normal tubular structure and,
instead, were more fragmented (Fig. 3D). To
examine how proximal this morphological defect
is to SRP depletion, we imaged cells at various
time points after auxin treatment (Fig. 4A and
fig. S7A) and categorized mitochondrial mor-
phology (fig. S7B). Mitochondrial morphology
defects were observed at early time points (Fig. 4B),
before detection of ER stress (fig. S7C) and while
ER morphology appeared normal (fig. S8). The
rapid emergence of mitochondrial morphology
defects in cells suggests that they are not a sec-
ondary consequence of ER morphological de-
fects; however, it is possible that the later, severe
mitochondrial defects are affected by the ER de-
fects. The observed mitochondrial fragmentation
required synthesis of new proteins, because cells
depleted of SRP and treated with CHX possessed
predominantly tubular mitochondria (fig. S9).
Thus, mistargeting of SRP-dependent proteins to
mitochondria disrupts mitochondrial structure.
Three important principles of SRP action now
emerge (Fig. 4C). First, a prominent subset of
proteins with cleavable N-terminal signal pep-
tides is efficiently cotranslationally targeted to
the ER in the complete and extended absence
of SRP. Second, with the exception of proteins
only containing TMDs at or near to the C ter-
minus (13–15), targeting and translocation of
proteins with anchoring TMDs, regardless of
their position relative to the N terminus, are
fully dependent on SRP. Together with work in
bacteria (8), our findings suggest that a univer-
sal feature of SRP’s action is to engage TMDs
across the length of the nascent chain. Third,
our results reveal an unanticipated role for SRP
in maintaining the specificity of organelle target-
ing. Without SRP, a subset of proteins becomes
susceptible to aberrant cotranslational targeting
to mitochondria reminiscent of the mistargeting
of some TA proteins to mitochondria in the ab-
sence of Get3/TRC40 ER targeting factors (13).
This may reflect a more general tendency of hy-
drophobic or aggregation-prone proteins to be
recruited to mitochondria (20). Mistargeting in
the absence of SRP triggered rapid mitochondrial
fragmentation, an indicator of mitochondrial
dysfunction. Consistent with SRP playing a
role in maintaining mitochondrial integrity,
yeast adapted for SRP deletions acquire rho–
(respiration-deficient) phenotypes (21, 22). This
role of SRP parallels that of NAC, which can
prevent mistargeting of mitochondrial proteins
to the ER in metazoans (11). Together, these ob-
servations illustrate the critical role of maintain-
ing specificity of protein targeting for proper
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We thank members of the Weissman and Nunnari labs for
discussion; C. Jan, M. Jost, and M. Shurtleff for critical reading of
the manuscript; and the University of California, San Francisco,
Center for Advanced Technology for technical assistance. This
work was supported by the Howard Hughes Medical Institute
(J.S. W.) and NIH grants R01AG041826 (J.S. W.), P50GM102706
(J.S. W.), R01GM062942 (J.N.), R01GM097432 (J.N.), and
R01GM106019 (J.N.). E.A.C. was supported by NSF Graduate
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Materials and Methods
Figs. S1 to S9
Tables S1 to S3
30 October 2017; accepted 4 January 2018
Published online 18 January 2018
692 9 FEBRUARY 2018 • VOL 359 ISSUE 6376 sciencemag.org SCIENCE
Auxin: 0 min 10 min 20 min 30 min 45 min 1 hr 1.5 hr 3 hr
+ SRP - SRP
Fig. 4. Loss of SRP leads to rapid mitochondrial defects. (A) Confocal fluorescence
microscopy of mitochondrial and ER morphology with Srp72-AID after indicated times of
auxin treatment. Mitochondrial Su9-TagBFP is shown as a maximum intensity projection
(MIP, top) or a single plane (blue, below) with ER–mCherry-HDEL (red). Scale bar, 2 mm.
(B) Quantification of mitochondrial morphologies from (A). (C) Summary of physiological
consequences of loss of SRP. Left: In the presence of SRP (orange rod), ribosomes translating
SRP-independent (purple) and -dependent (orange) proteins are targeted to the ER. Right:
Without SRP, SRP-independent proteins are targeted to the ER, and proteins not targeted to
the ER can form aggregates (top) or be mistargeted to mitochondria (pink), leading to