Download Roles of Arabidopsis PARC6 in Coordination of

Roles of Arabidopsis PARC6 in Coordination of the
Chloroplast Division Complex and Negative
Regulation of FtsZ Assembly1[OPEN]
Min Zhang 2, Cheng Chen 2, John E. Froehlich, Allan D. TerBush, and Katherine W. Osteryoung *
Department of Plant Biology (M.Z., C.C., A.D.T., K.W.O.), Michigan State University-Department of Energy
Plant Research Laboratory (J.E.F.), and Department of Biochemistry and Molecular Biology (J.E.F.), Michigan
State University, East Lansing, Michigan 48824; and College of Life Sciences, Capital Normal University,
Beijing 100048, China (M.Z.)
ORCID IDs: 0000-0002-3716-4565 (M.Z.); 0000-0002-0028-2509 (K.W.O.).
Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The
assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope
membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that
PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream
of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog
PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However,
whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here,
we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain
and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct
inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in
Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ
assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2
interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of
PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates
the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.
Chloroplasts evolved from an ancient cyanobacterium through endosymbiosis (Gould et al., 2008;
Keeling, 2013). Like their prokaryotic relatives, chloroplasts replicate by binary fission, which is driven by a
dynamic macromolecular complex located at the middle of the organelle (Falconet, 2011; Miyagishima et al.,
2011; Osteryoung and Pyke, 2014). The major contractile
components of the division complex include the FtsZ
1
This work was supported by the U.S. Department of Energy,
Office of Science, Basic Energy Sciences (grant nos. DE–FG02–
06ER15808 to K.W.O. and DE–FG02–91ER20021 to J.E.F.), the
National Natural Science Foundation of China (grant no. 31470296
to M.Z.), and the U.S. National Science Foundation (grant no. 1121943
to K.W.O.).
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Katherine W. Osteryoung ([email protected]).
M.Z. and K.W.O. conceived and designed the research plans; M.Z.,
C.C., J.E.F., and A.D.T. performed all experiments and analyzed data;
M.Z., C.C., and K.W.O wrote the article with contributions from all
authors. K.W.O. supervised the research and completed the writing.
[OPEN]
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250
ring (Z ring), which assembles on the stromal surface of
the inner envelope membrane (IEM; McAndrew et al.,
2001; Vitha et al., 2001), and the DYNAMIN-RELATED
PROTEIN 5B (DRP5B; also called ACCUMULATION
AND REPLICATION OF CHLOROPLASTS5 [ARC5])
ring, which assembles on the cytosolic surface of the
outer envelope membrane (OEM; Gao et al., 2003;
Miyagishima et al., 2003; Yoshida et al., 2006). In green
algae and land plants, the Z ring is composed of the
tubulin-like, heteropolymer-forming proteins FtsZ1
and FtsZ2, which are both required for normal Z-ring
function (Schmitz et al., 2009; TerBush and Osteryoung,
2012). DRP5B is a member of the dynamin family of
membrane fission proteins, which polymerize into
collar-like structures to mediate a variety of membrane
fission processes in eukaryotes (Morlot and Roux,
2013). The Z ring and DRP5B ring function together
to drive the simultaneous constriction of the IEM and
OEM during chloroplast division.
The assembly and constriction of the inner Z ring and
outer DRP5B ring are coordinated across the two
membranes by the activities of midplastid-localized
envelope membrane proteins whose functions have
been studied in Arabidopsis (Arabidopsis thaliana).
ARC6 (Pyke et al., 1994) is a bitopic IEM protein of
cyanobacterial origin that is conserved throughout
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Role of PARC6 in Chloroplast Division
green-lineage chloroplasts (Koksharova and Wolk,
2002; Vitha et al., 2003; Osteryoung and Pyke, 2014). Its
N-terminal region extends into the stroma, where it
interacts directly and specifically with FtsZ2 (Maple
et al., 2005). As FtsZ1 and FtsZ2 are soluble (McAndrew
et al., 2001), this interaction probably serves both to
tether the Z ring to the IEM and to promote FtsZ polymerization at the division site (Vitha et al., 2003). The
C-terminal region of ARC6 protrudes into the intermembrane space (IMS) and interacts with the IMS region
of the plant-specific bitopic OEM protein PLASTID
DIVISION2 (PDV2). ARC6-PDV2 interaction is required
for the localization of PDV2 to the midplastid (Glynn
et al., 2008). PDV2 and its paralog PDV1, also in the
OEM, in turn recruit DRP5B from a cytosolic pool to the
OEM (Miyagishima et al., 2006), probably through direct
interaction with their cytosolic regions (Holtsmark et al.,
2013). Thus, interactions between FtsZ2 and ARC6 in the
stroma, ARC6 and PDV2 in the IMS, and PDV2 (and
PDV1) and DRP5B in the cytosol connect and coordinate
the FtsZ and DRPB5B rings across the IEM and OEM.
Previously, we showed that, despite the fact that an
interaction between the IMS regions of ARC6 and PDV1
could not be detected, ARC6 was nevertheless required
for the equatorial localization of PDV1 as well as PDV2,
suggesting the existence of a factor that acted downstream of ARC6 to position PDV1 (Glynn et al., 2008).
This downstream factor was subsequently shown to
be the nucleus-encoded chloroplast division protein
PARALOG OF ARC6 (PARC6; Glynn et al., 2009), also
called CDP1 (Zhang et al., 2009) and ARC6H (Ottesen
et al., 2010). parc6 mutants exhibited mislocalization of
PDV1 but not PDV2, demonstrating a specific role for
PARC6 in PDV1 positioning. PARC6 is restricted to
vascular plants, suggesting that it arose by the duplication and divergence of ARC6 following separation of the
nonvascular and vascular lineages. As suggested by its
name, PARC6 shares significant sequence similarity
with ARC6 and is similarly imported to the chloroplast
by a cleavable N-terminal transit peptide and localized
in the IEM. However, whereas ARC6 has a single
transmembrane domain (TMD), PARC6 is predicted to
bear two, and while a portion of its N terminus was
clearly shown to reside in the stroma, its full topology
has not been established (Glynn et al., 2009). Furthermore, genetic analysis suggested that, unlike ARC6,
which positively regulates FtsZ assembly (Vitha et al.,
2003), PARC6 functions partly as a negative regulator of
FtsZ assembly. Interaction assays provided evidence
that this negative regulation may be mediated by interaction of the N terminus of PARC6 with the stromal
division protein ARC3 (Pyke et al., 1994; Shimada et al.,
2004; Maple et al., 2007), a Z-ring positioning factor recently shown to inhibit Z-ring assembly and/or promote
FtsZ filament and Z-ring destabilization (TerBush and
Osteryoung, 2012; Zhang et al., 2013; Johnson et al.,
2015). Although the interaction of PARC6 with FtsZ was
not detected previously, the significance of this finding
has remained uncertain in the absence of definitive data
on PARC6 topology (Glynn et al., 2009).
Here, we report a detailed topological analysis of
Arabidopsis PARC6, investigate its interactions with
other division factors, and assess the effect of PARC6 on
chloroplast FtsZ assembly. Our findings provide evidence that the negative effect of PARC6 on Z-ring assembly results from its interaction with ARC3 and
reveal a role for PARC6 in coordinating the inner Z ring
and outer DRP5B ring partially analogous to the role of
ARC6.
RESULTS
PARC6 Topology Resembles That of ARC6
As described previously (Glynn et al., 2009), PARC6
(At3g19180) is predicted to bear two TMDs: TMD1
(amino acids 357–377) and TMD2 (amino acids 574–
596; Fig. 1A, top). To analyze PARC6 topology, we
carried out in vitro chloroplast import and fractionation
assays on wild-type PARC6 from Arabidopsis and on a
series of PARC6 variants in which TMD1, TMD2, or
both were deleted (Fig. 1A). Radiolabeled precursor
proteins (labeled pr in Fig. 1B) produced by in vitro
translation of full-length PARC6 and the TMD deletion
constructs migrated at approximately their predicted
masses on SDS-PAGE gels (Fig. 1B, lane 1). When incubated with isolated chloroplasts, a significant fraction
of the radiolabeled proteins migrated at a lower mass
(Fig. 1B, lanes 2 and 3, labeled m), indicating protein
import and transit peptide processing to yield mature
protein. The smaller PARC6 and PARC6(DTMD1) import products were enriched in the membrane fraction
(Fig. 1B, lane 2, arrows), while the PARC6(DTMD2) and
PARC6(DTMD1/2) import products were enriched in
the soluble fractions (Fig. 1B, lane 3, arrowheads). These
results indicate that the association of PARC6 with the
membrane fraction is mediated solely by TMD2, implying that TMD2 is the only authentic TMD in PARC6
(asterisks in Fig. 1A).
Because PARC6 and ARC6 are paralogous proteins
and ARC6 also has a single TMD roughly corresponding to that of PARC6 TMD2 (Vitha et al., 2003; Glynn
et al., 2009; Fig. 1A, bottom), the above results suggested that Arabidopsis PARC6 is a bitopic IEM protein
with a topology similar to that of ARC6 (Vitha et al.,
2003): that is, with the region upstream of the authentic
TMD (TMD2) oriented toward the stroma and the region downstream of this TMD situated in the IMS. To
test these predictions, we conducted protease protection assays on the radiolabeled PARC6 import product
(Fig. 1C, top). Following import reactions performed as
described above, chloroplasts were treated either with
thermolysin, which does not penetrate the OEM and
therefore only degrades regions of proteins exposed to
the chloroplast exterior, or with trypsin, which penetrates the OEM but not the IEM and therefore degrades
regions of proteins exposed to both the chloroplast exterior and the IMS but not the stroma (Cline et al., 1984;
Jackson et al., 1998). The PARC6 import product was
protected from degradation by thermolysin (Fig. 1C,
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Zhang et al.
top, lane 4). In contrast, trypsin treatment produced a
smaller radiolabeled fragment that was associated with
the membrane fraction (Fig. 1C, top, lane 6, asterisk). In
control assays, a stromal protein was protected from
both trypsin and thermolysin treatment and was
enriched in the soluble fraction (Fig. 1C, bottom). Although proteolysis by trypsin was incomplete, the size
of the trypsin-protected PARC6 fragment (Fig. 1C, top,
asterisk) was consistent with the calculated molecular
mass (57.3 kD) of the region of PARC6 between the end
of the predicted transit peptide and the end of TMD2
(amino acids 77–596; Fig. 1A, top). These PARC6 fractionation and protease-protection patterns closely resembled those of ARC6 (Fig. 1, B, bottom, and C,
middle; Vitha et al., 2003). Based on the combined data,
we conclude that, similar to ARC6 (Vitha et al., 2003),
Arabidopsis PARC6 is a bitopic IEM protein with its
larger N-terminal region (amino acids 77–573) exposed
to the stroma and its smaller C-terminal region (amino
acids 597–819) exposed to the IMS.
The Stromal Region of PARC6 Interacts with the
Membrane Occupation and Recognition Nexus Domain
of ARC3
Previously, we assessed amino acids 77 to 356 of
PARC6 (PARC677-356) for interaction with the stromal
chloroplast division proteins FtsZ1, FtsZ2, and ARC3
based on evidence from pea (Pisum sativum) that this
region of PARC6 resides in the stroma. Only ARC3 was
shown to interact (Glynn et al., 2009). Based on the topological analysis described above, we revisited these
interactions by using the newly defined full-length
stromal region of PARC6 (PARC677-573) in yeast twohybrid assays. As shown in Figure 2, both full-length
ARC3 (lacking its predicted transit peptide) and a
Figure 1. Topological analysis of Arabidopsis PARC6 at the IEM. A,
Diagrams of PARC6 and ARC6 constructs used for chloroplast import
assays. TP, Transit peptide; TMD, predicted TMD; *, authentic PARC6
TMD; AA, amino acids. B, In vitro [35S]PARC6, [35S]PARC6(DTMD1),
[35S]PARC6(DTMD1/2), [35S]PARC6(DTMD2), and [35S]ARC6 translation products (Tr, 10% of each translation reaction loaded; lane 1) were
incubated with isolated intact pea chloroplasts for 30 min (Import; lanes 2
and 3). After import, intact chloroplasts were recovered by centrifugation,
lysed, and fractionated into total membrane (P; lane 2) or soluble (S; lane
3) fractions. All fractions were then analyzed by SDS-PAGE and fluorography. MM, Molecular mass markers; pr, precursor protein; m, mature
import product. White arrows and arrowheads highlight the association of
the import products with the membrane and soluble fractions, respectively. Lower bands in lane 1 likely resulted from initiation at internal Met
codons during in vitro translation reactions (Teng et al., 2012). Where
present, dark upper bands in lane 2 represent unimported precursor
protein that remained associated with chloroplasts following import
and recovery. C, Protease protection assays. In vitro [35S]PARC6 (top),
[35S]ARC6 (middle), and [35S]SSU (Rubisco small subunit; bottom) translation products (Tr, 10% of each translation reaction loaded; lane 1) were
incubated with isolated intact pea chloroplasts for 30 min (Import; lanes 2–
7). Reactions were then treated with (+) or without (2) either thermolysin
or trypsin. Intact chloroplasts were recovered by centrifugation, lysed, and
fractionated into total membrane (lanes 2, 4, and 6) or soluble (lanes 3, 5,
and 7) fractions. All fractions were analyzed by SDS-PAGE and fluorography. *, Trypsin-protected fragments of PARC6 (top) and ARC6 (middle).
252
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Role of PARC6 in Chloroplast Division
The Stromal Region of PARC6 Interacts with FtsZ2
Figure 2. Yeast two-hybrid assays between PARC6 stromal regions
and the stromal chloroplast division proteins FtsZ1, FtsZ2, and ARC3.
Growth of Y2HGold cells was assayed in the presence (His) or absence
(2His) of His to detect the activation of the HIS3 reporter. The latter
medium was supplemented with 0.1 mg mL21 of the toxic drug aureobasidin A (AbA) to detect two-hybrid interactions based on activation of
the AUR1-C reporter, which confers aureobasidin A resistance in the
Y2HGold strain. Constructs were expressed in the pGAD-T7 (AD) or
pGBK-T7 (BD) vector as indicated. Empty vectors were used as controls.
Dilutions from the same starting culture are indicated at bottom. Predicted transit peptides were excluded from all constructs. Construct and
strain details are described in “Materials and Methods.”
C-terminal region of ARC3 bearing the membrane
occupation and recognition nexus (MORN) motifs
(Shimada et al., 2004), referred to as the MORN domain
(amino acids 598–741; Maple et al., 2007), interacted
with PARC677-573 (Fig. 2, rows 3 and 5). Deletion of the
MORN domain from ARC3 abolished the interaction
(Fig. 2, row 4). As with full-length ARC3 (Glynn et al.,
2009), the ARC3 MORN domain alone also interacted
with PARC677-356 (Fig. 2, row 17). These findings confirm that PARC6 interacts with ARC3 in yeast twohybrid assays and indicate that amino acids 77 to 356
of the newly defined PARC6 stromal region is sufficient
for this interaction. These results further suggest that
the PARC6-ARC3 interaction may be mediated solely
by the MORN-containing C-terminal region of ARC3.
We also tested whether PARC677-573 interacts with
FtsZ1 and FtsZ2. As observed for PARC677-356 (Glynn
et al., 2009), no interaction between FtsZ1 and PARC677-573
was detected (Fig. 2, row 1). In contrast, PARC677-573 exhibited a specific interaction with FtsZ2 (Fig. 2, row 2).
Although the latter results suggested that PARC6357-573
could be responsible for the PARC6 interaction with
FtsZ2, this region by itself did not support the interaction
(Fig. 2, row 13), suggesting that the full-length stromal
region of PARC6 or a region overlapping with both
PARC677-356 and PARC6357-573 is required.
To further analyze the interactions between PARC6
and the FtsZ proteins, we fused mCherry to the stromal
region of PARC6 to create PARC677-573-mCherry and
coexpressed it with either FtsZ1-eYFP (enhanced yellow fluorescent protein) or FtsZ2-eCFP (enhanced cyan
fluorescent protein) in the fission yeast Schizosaccharomyces pombe. Recently, S. pombe has emerged as a valuable heterologous system in which to analyze the
behavior of bacterial and chloroplast FtsZ filaments as
well as to test the interactions with and effects of putative FtsZ assembly regulators (Srinivasan et al., 2007,
2008; TerBush and Osteryoung, 2012; Zhang et al.,
2013). When expressed alone in S. pombe, PARC677-573mCherry fluorescence appeared diffusely localized
in the cytosol (Fig. 3A), similar to the localization of
unfused mCherry (Fig. 3B). As shown previously
(TerBush and Osteryoung, 2012), FtsZ1-eYFP and FtsZ2eCFP assembled into solid cables and more elaborate
filament networks, respectively (Fig. 3, C and D).
In coexpression strains, PARC677-573-mCherry colocalized with FtsZ2-eCFP to a filament network similar
to that formed by FtsZ2-eCFP alone (Fig. 3, D and E).
We used Pearson’s correlation coefficient (PCC) to
quantify the extent of overlap between the two fluorescence signals and how closely the signal intensities
correlate (Bolte and Cordelières, 2006). The PARC677-573mCherry and FtsZ2-eCFP fluorescence signals had a
PCC of 0.61 6 0.04 (mean 6 SE; n = 9 cells), indicating that
these proteins colocalize within the filament network
and that their signal intensities are directly proportional
to each other (i.e. as one signal increases, so does the
other). As a control, unfused mCherry localized diffusely
when coexpressed with FtsZ2-eCFP (Fig. 3F), and the
PCC between fluorescence signals was 0.28 6 0.05 (n =
10), indicating a low degree of colocalization. The difference in colocalization was also visually evident from
the images showing the merged fluorescence signals
(Fig. 3, E and F, merge). Notably, PARC677-573-mCherry
did not appear to affect the formation of the FtsZ2eCFP filament network (Fig. 3, D and E), unlike
ARC3, which inhibits FtsZ2 (and FtsZ1) filament assembly in S. pombe (TerBush and Osteryoung, 2012;
Zhang et al., 2013). In contrast with FtsZ2-eCFP,
FtsZ1-eYFP exhibited a very low degree of colocalization with PARC677-573-mCherry (Fig. 3G; PCC of
0.14 6 0.05 [n = 11]), consistent with yeast two-hybrid
results (Fig. 2, row 1).
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Zhang et al.
Figure 3. The stromal region of PARC6 colocalizes with FtsZ2 filaments in S. pombe. A to D, Epifluorescence and differential
interference contrast (DIC) micrographs of cells expressing PARC677-573-mCherry (A), mCherry (B), FtsZ1-eYFP (C), and FtsZ2eCFP (D). E to G, Fluorescence imaging of cells coexpressing PARC677-573-mCherry and FtsZ2-eCFP (E), mCherry and FtsZ2eCFP (F), and PARC677-573-mCherry and FtsZ1-eYFP (G). eYFP and eCFP signals are falsely colored green; mCherry signals are
falsely colored magenta. White areas in the merged images represent regions of signal overlap. Dotted lines indicate cell
outlines. Bars = 5 mm.
FtsZ2 interacts with ARC6 through a C-terminal
peptide conserved among bacterial FtsZ and chloroplast FtsZ2 proteins (Vaughan et al., 2004; Maple et al.,
2005). Since PARC6 is a paralog of ARC6, we tested
whether the C-terminal tail of FtsZ2 mediates its interaction with PARC6 in yeast two-hybrid assays. Like
ARC6 (Fig. 4A, rows 5 and 6), PARC677-573 interacted
with the presumed mature form of FtsZ2 lacking
its predicted transit peptide (FtsZ249-478; Olson et al.,
2010; Fig. 4A, row 1) but not with a form lacking the
C-terminal 18 amino acids (FtsZ249-460) that includes the
conserved C-terminal peptide mentioned above (amino
acids 463–478; Schmitz et al., 2009; Fig. 4A, row 2). In
contrast to a previous report (Maple et al., 2005), mutation of a conserved Phe in this region (Phe-466) to Ala
did not completely abolish the interaction of FtsZ2 with
ARC6 (Fig. 4A, row 7). However, the FtsZ2-PARC6
interaction was abolished by the FtsZ2F466A mutation
in this assay (Fig. 4A, row 3).
To further evaluate these findings, we coexpressed
three different versions of FtsZ2 that were C-terminally
fused to the monomeric fluorescent protein mCerulean
(Shaner et al., 2004; Papapetrou et al., 2009) in S. pombe
and assessed the extent of their colocalization with
PARC677-573-mCherry. When expressed alone, FtsZ249-478mCerulean and FtsZ2F466A-mCerulean assembled large
ring-shaped structures, likely composed of loosely bundled FtsZ2 polymers (TerBush and Osteryoung, 2012;
Fig. 4B, top and bottom), while FtsZ249-457-mCerulean,
which lacked the C-terminal 21 amino acids of FtsZ2,
assembled smaller rings and more complex networks
(Fig. 4B, middle). The difference in the morphology of
filaments formed by FtsZ249-478-mCerulean (Fig. 4B, top)
and FtsZ2-eCFP (Fig. 3D) in S. pombe is likely due to the
monomeric nature of mCerulean, as eCFP retains the
intact dimer interface (Zacharias et al., 2002).
In coexpression strains, PARC677-573-mCherry colocalized with FtsZ2 49-478-mCerulean with a PCC of
0.64 6 0.02 (n = 21; Fig. 4C, top row; Supplemental Fig.
S1A). When coexpressed with FtsZ249-457-mCerulean,
PARC677-573-mCherry localized much more diffusely
(Fig. 4C, middle row) and exhibited a reduced degree
of colocalization with FtsZ249-457-mCerulean (PCC of
0.53 6 0.05 [n = 23]) compared with its colocalization
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Role of PARC6 in Chloroplast Division
with FtsZ249-478-mCerulean (P = 0.056). However, a
small proportion of cells still showed weak colocalization between PARC677-573-mCherry and FtsZ249-457mCerulean (Fig. 4C, middle row; Supplemental Fig.
S1B, top row), suggesting that the interaction was not
completely abolished. Colocalization of PARC677-573mCherry with FtsZ2F466A-mCerulean was also reduced
(PCC of 0.37 6 0.05 [n = 17, P = 0.000]; Fig. 4C, bottom
row; Supplemental Fig. S1C). The higher PCC value
for the colocalization of PARC677-573-mCherry with
FtsZ249-457-mCerulean than FtsZ2F466A-mCerulean may
reflect the more extensive FtsZ2 filament network
formed by the former, resulting in a higher degree of
signal overlap with PARC677-573-mCherry. In control
experiments, ARC668-614-mVenus, bearing the stromal
region of ARC6 (Fig. 1A, bottom; Vitha et al., 2003), also
had a significantly lower affinity for FtsZ249-457-mCerulean
(PCC of 0.6 6 0.04 [n = 16]) and FtsZ2F466A-mCerulean
(PCC of 0.51 6 0.04 [n = 21]) than FtsZ2 49-478 mCerulean (PCC of 0.8 6 0.03 [n = 14]) in S. pombe
(Fig. 4E).
Together with the yeast two-hybrid results, our
colocalization data in S. pombe indicate that the stromal
region of PARC6 interacts with FtsZ2 but not FtsZ1 and
that the C-terminal tail of FtsZ2, including the conserved Phe, at least partly mediates this interaction,
similar to the interaction between ARC6 and FtsZ2.
Overexpression of PARC6 Inhibits FtsZ Assembly in
Transgenic Plants
Previously, we showed that FtsZ forms abnormally
long filaments in Arabidopsis parc6-1 mutants, suggesting that PARC6 functions as a negative regulator of
Z-ring assembly in wild-type plants (Glynn et al., 2009).
To further investigate the role of PARC6 in chloroplast
division, we generated a construct encoding a C-terminally
tagged PARC6-Myc fusion protein for overexpression
in Arabidopsis. To confirm functionality, we first
expressed PARC6-Myc from the native PARC6 promoter
Figure 4. Effect of the conserved C-terminal tail of FtsZ2 on interaction
with PARC6. A, Yeast two-hybrid assays between the indicated forms of
FtsZ2 and the stromal region of PARC6 (PARC677-573) or a fragment of
ARC6 (ARC6154-509) shown previously to interact with FtsZ2 (Glynn
et al., 2009). Growth of Y2HGold cells was assayed in the presence
(+His) or absence (2His) of His to detect the activation of the HIS3
reporter. The latter medium was supplemented with 0.1 mg mL21 of the
toxic drug aureobasidin A (AbA) to detect two-hybrid interactions based
on the activation of the AUR1-C reporter, which confers aureobasidin A
resistance in the Y2HGold strain. Constructs were expressed in the
pGAD-T7 (AD) or pGBK-T7 (BD) vector as indicated. Empty vectors
were used as controls. Dilutions from the same starting culture are
indicated at bottom. B to E, Epifluorescence micrographs of cells
expressing FtsZ249-478-mCerulean (top), FtsZ249-457-mCerulean (middle), or FtsZ2F466A-mCerulean (bottom; B); PARC677-573-mCherry and
FtsZ249-478-mCerulean (top), PARC677-573-mCherry and FtsZ249-457mCerulean (middle), or PARC677-573-mCherry and FtsZ2F466A-mCerulean
(bottom; C); ARC668-614-mVenus (D); or ARC668-614-mVenus and FtsZ249-478mCerulean (top), ARC668-614-mVenus and FtsZ249-457-mCerulean (middle),
or ARC668-614-mVenus and FtsZ2F466A-mCerulean (bottom; E). mCherry
(mCh) and mVenus (mVe) fluorescence signals are falsely colored magenta; mCerulean (mCer) fluorescence signals are falsely colored green.
Dotted lines indicate cell outlines. Bars = 5 mm.
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in the parc6-1 knockout mutant (SALK_100009), which
exhibits enlarged chloroplasts with heterogenous morphologies and abnormal Z rings and filaments (described below; Fig. 5A; Supplemental Fig. S2A; Glynn
et al., 2009). Following selection, leaf tissue from the
transgenic plants was fixed and mesophyll cells were
observed microscopically. The fusion protein restored
both normal chloroplast size and number and the localization of FtsZ to single midplastid rings when
expressed at wild-type levels (Supplemental Fig. S2, A
and B), demonstrating that it retained functionality.
For overexpression studies, we expressed PARC6Myc under the control of the cauliflower mosaic virus
35S promoter in wild-type Columbia-0 plants. Transgenic lines contained chloroplasts that were greatly
enlarged and heterogenous in size compared with those
in the wild type, and the number of chloroplasts was
significantly reduced (Fig. 5A), indicating severe impairment of chloroplast division. Although these chloroplast morphology phenotypes resembled those of the
parc6-1 mutant, immunoblotting with an anti-PARC6
antibody confirmed significant accumulation of the
PARC6 fusion protein in these plants (Fig. 5B), indicating that overexpression of PARC6 inhibits chloroplast division.
Immunofluorescence staining with antibodies against
FtsZ1 and FtsZ2-1 was conducted to determine the effect of PARC6 overexpression on FtsZ assembly in
plants. Midplastid-localized Z rings were detected in
the wild type, whereas multiple misplaced and long
FtsZ filaments were observed in parc6-1 mutants (Fig.
5A; Supplemental Fig. S2A), as reported previously
(Glynn et al., 2009). In contrast, in transgenic lines with
high levels of PARC6-Myc, FtsZ appeared to localize to
short and disorganized filaments (Fig. 5A), suggesting
a disruption of FtsZ assembly. This phenotype was not
caused by altered FtsZ accumulation in these plants,
because FtsZ1 and FtsZ2 protein levels were comparable to those in wild-type plants (Fig. 5B). Together,
these findings reveal that overexpression of PARC6
inhibits FtsZ assembly and thus interferes with chloroplast division, consistent with parc6-1 mutant analysis
suggesting that PARC6 negatively regulates assembly
(Glynn et al., 2009). However, our finding that the stromal region of PARC6 does not inhibit FtsZ2 assembly
in S. pombe cells (Fig. 3E) suggests that this inhibition is
indirect.
The IMS Regions of PARC6 and PDV1 Interact
The homologous chloroplast division proteins PDV1
and PDV2 both reside in the OEM and have single
TMDs and defined topologies (Miyagishima et al., 2006;
Glynn et al., 2008). PDV1 has been shown to localize to
the middle of deeply constricted chloroplasts and to a
single spot at one pole following division, resembling
the localization pattern of PARC6. Furthermore, genetic
analysis has shown that PARC6 is required for the localization of PDV1 but not PDV2 to the chloroplast
Figure 5. Overexpression of PARC6 disrupts Z-ring assembly in transgenic plants. A, Chloroplast phenotype images (differential interference
contrast [DIC]) and immunofluorescence localization of FtsZ in mesophyll
cells of the indicated genotypes. WT + 35S::PARC6-Myc, Wild-type (WT)
Columbia-0 plants transformed with the 35S::PARC6-Myc construct. FtsZ1
and FtsZ2 were immunolabeled with anti-FtsZ1 and anti-FtsZ2-1 antibodies, as indicated. Green, Alexa Fluor 488-labeled goat anti-rabbit
secondary antibody for anti-FtsZ1 and anti-FtsZ2-1 antibodies; magenta,
chlorophyll autofluorescence. Bars are as indicated. B, Immunoblot
analysis of PARC6, FtsZ1, and FtsZ2-1 in the indicated plants. Total proteins extracted from 5 mg of 9-d-old seedlings were loaded in each lane.
Ponceau S-stained Rubisco large subunit (bottom) served as a loading
control. Molecular mass markers (MM) are indicated on the right.
division site (Glynn et al., 2009). We performed yeast
two-hybrid assays to determine whether the IMS region
of PDV1 (PDV1IMS) or PDV2 (PDV2IMS) would interact
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Role of PARC6 in Chloroplast Division
with the newly defined IMS region of PARC6 (PARC6IMS)
described above (Fig. 1A). PARC6IMS interacted with
PDV1IMS but not PDV2IMS (Fig. 6A, rows 4 and 5). To
further verify the interaction with PDV1IMS, we performed in vitro pull-down assays. Maltose-binding
protein (MBP)-tagged PARC6IMS expressed in Escherichia
coli could be precipitated from crude E. coli extracts by
glutathione-Sepharose beads coated with glutathione
S-transferase (GST)-tagged PDV1IMS but not by GSTcoated or empty beads (Fig. 6B, lanes 1–3).
Both PDV1 and PDV2 bear conserved Gly residues at
their C termini. One of the alleles that led to the identification of PDV1 and PDV2 as chloroplast division
proteins by mutant screening (pdv1-2) resulted from
mutation of the PDV1 C-terminal Gly (Gly-272) to Asp,
and PDV1 was mislocalized in this mutant (Miyagishima
et al., 2006). A similar mutation in PDV2 greatly diminished its unique interaction with ARC6 (Glynn et al.,
2008). To test whether the C-terminal Gly in PDV1 is
likewise important for its interaction with PARC6, we
generated PDV1IMS(G272D) constructs for use in both yeast
two-hybrid and pull-down assays. PDV1IMS(G272D) failed
to interact with PARC6IMS in yeast (Fig. 6A, row 7),
and the same mutation significantly attenuated the
binding of the PDV1 IMS region to MBP-PARC6IMS in
pull-down experiments (Fig. 6B, lane 4). Together, these
results strongly suggest that PARC6-dependent localization of PDV1 to the chloroplast division site is mediated by their direct interaction in the IMS and that this
interaction is dependent on the C-terminal Gly in PDV1.
DISCUSSION
Figure 6. The IMS regions of PARC6 and PDV1 interact. A, Yeast twohybrid assay between the IMS regions of PARC6 and the PDV proteins.
Growth of AH109 cells was assayed in the presence (+His) or absence
(2His) of His to detect the activation of the HIS3 reporter. The latter
medium was supplemented with 2.5 mM 3-amino-1,2,4-triazole (3-AT).
Dilutions from the same starting culture are indicated at bottom. The
PDV1IMS and PDV2IMS constructs are described by Glynn et al. (2008).
The PDV1(G272D) construct is described in the text. Constructs were
expressed in the pGAD-T7 (AD) or pGBK-T7 (BD) vector as indicated.
B, In vitro pull-down assay of PARC6IMS and PDV1IMS. GlutathioneSepharose 4B beads were treated with buffer only (lane 1) or coated
with GST (lane 2), GST-tagged PDV1IMS (lane 3), or PDV1IMS(G272D) (lane
4). The beads were then incubated with crude extracts from E. coli cells
expressing MBP-PARC6IMS. Protein was eluted and analyzed by immunoblotting with anti-MBP and anti-GST antibodies. Input, Ten percent of MBP-PARC6IMS extract added to pull-down assays.
A previous in vivo analysis of a PARC6 homolog in
pea yielded only a partial model of PARC6 topology,
which indicated that the region between the predicted
transit peptide and the predicted TMD1 (Fig. 1A) was in
the stromal compartment (Glynn et al., 2009). In vitro
chloroplast import and protease protection assays
performed here on Arabidopsis PARC6 and its TMDdeleted derivatives have now revealed that TMD2 is the
only authentic TMD in PARC6 and that the previously
predicted TMD1 is, in fact, part of the stromal region of
PARC6. Therefore, PARC6 is a bitopic IEM protein in
which the longer N-terminal region faces the stroma
and the shorter C-terminal region protrudes into the
IMS (Fig. 7). This topology is equivalent to that of ARC6
(Vitha et al., 2003).
Both PARC6 and ARC3 function as negative regulators of FtsZ assembly, as indicated by the presence of
excessively long FtsZ filaments and multiple Z rings in
parc6-1 and arc3 mutants, respectively (Fig. 5; Glynn
et al., 2009; Zhang et al., 2013). Consistent with these
phenotypes, overexpression of both PARC6 and ARC3
in vivo had a similar inhibitory effect on FtsZ assembly
and chloroplast division (Fig. 5; Zhang et al., 2009).
However, PARC6 did not inhibit FtsZ2 assembly in
S. pombe (Figs. 3E and 4), whereas ARC3 did (Zhang
et al., 2013). Collectively, these findings indicate that the
role of PARC6 as a negative regulator of FtsZ assembly
in vivo is an indirect effect of its interaction with ARC3.
Interestingly, the localization of FtsZ in PARC6 overexpression lines (Fig. 5) resembled that of a PARC6GFP fusion protein overexpressed in wild-type plants
(Zhang et al., 2009), consistent with our data showing
PARC6-FtsZ2 colocalization in S. pombe (Figs. 3E and 4).
Our interaction assays based on the newly determined PARC6 topology demonstrated that the stromal
region of PARC6 interacts not only with ARC3, as
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Zhang et al.
Figure 7. Working model of the chloroplast division complex emphasizing the roles of PARC6 and ARC6. A, The topology of
PARC6 in the IEM is equivalent to that of ARC6 (i.e. it has a single transmembrane span with its N-terminal region in the stroma and its
C-terminal region in the IMS; Fig. 1). Z-ring assembly is restricted to the midplastid by the chloroplast Min system, which includes
ARC3 (Glynn et al., 2007; Maple et al., 2007; Zhang et al., 2013) as well as MinD1, MinE1, and MULTIPLE CHLOROPLAST
DIVISION SITE1 (not shown; Miyagishima et al., 2011; Osteryoung and Pyke, 2014). The Z ring, composed of FtsZ1/FtsZ2 heteropolymers that may assemble with mixed stoichiometries (Olson et al., 2010), is tethered to the IEM by ARC6 through direct
interaction with FtsZ2 in the stroma (Maple et al., 2005; Schmitz et al., 2009). ARC6 positions PDV2 to the division site in the OEM
through direct interaction of their IMS regions (Glynn et al., 2008). PARC6 functions similarly downstream of ARC6, interacting with
FtsZ2 in the stroma (Figs. 2–4) and positioning PDV1 at the division site (Glynn et al., 2009) through direct interaction in the IMS (Fig.
6). PDV1 and PDV2 independently recruit DRP5B from the cytosol to the OEM, but both PDV proteins are required for full DRP5B
contractile activity (Miyagishima et al., 2006), probably involving direct interactions between DRP5B and the PDV proteins and
between the cytosolic regions of PDV1 and PDV2 (Holtsmark et al., 2013). Thus, PARC6 and ARC6 coordinate the Z ring and
DRPB5B ring across the envelope membrane, enabling them to function together to constrict the envelope membranes. B, PARC6
recruits ARC3 to the division site, possibly during constriction. PARC6 binds to the MORN domain of ARC3 (Fig. 2; Glynn et al.,
2009), allowing ARC3, an FtsZ assembly inhibitor (Zhang et al., 2013), to interact with the Z ring. This interaction may facilitate
Z-ring remodeling and disassembly during constriction (Johnson et al., 2015). Other details omitted for simplicity are reviewed by
Miyagishima et al. (2011) and Osteryoung and Pyke (2014). C, C terminus; N, N terminus; Z1, FtsZ1; Z2, FtsZ2. Note that proteins
shown are not meant to represent stoichiometric ratios, as these have not been established.
shown previously (Glynn et al., 2009), but also with
FtsZ2 (Figs. 2 and 3). ARC3 prevents Z-ring misplacement in chloroplasts (Glynn et al., 2007; Maple et al.,
2007), very likely by directly inhibiting the formation
of Z rings distant from the midplastid division site,
perhaps by destabilizing the assembly of nascent FtsZ
polymers, although the mechanism remains unclear
(Zhang et al., 2013; Johnson et al., 2015). Although
ARC3 interacts with and inhibits the assembly of both
FtsZ1 and FtsZ2 filaments, because FtsZ2 imparts stability to the Z ring, we proposed that the ARC3-FtsZ2
interaction may be particularly important in preventing
Z-ring formation at nondivision sites (Zhang et al.,
2013). However, both PARC6 and ARC3 localize partly
to the division site (Shimada et al., 2004; Glynn et al.,
2009). Our findings that the N-terminal portion of the
PARC6 stromal region (PARC677-356) binds directly to
the ARC3 MORN domain (Fig. 2), and that this interaction requires the MORN domain (Glynn et al., 2009),
suggest that PARC6 may recruit ARC3 to the division
site via this domain. In contrast, the MORN domain
inhibits the interaction between ARC3 and FtsZ2 (and
FtsZ1; Zhang et al., 2013). Together, these data suggest
a model in which PARC6 may promote ARC3 activity
in vivo by sequestering the MORN domain, thus freeing
ARC3 to interact with FtsZ filaments (Fig. 7B). This
hypothesis will be tested in future experiments.
Although we do not yet know the significance of
ARC3 localization to the division site in wild-type
plants, it is possible that PARC6-ARC3-FtsZ2 interactions could promote Z-ring remodeling and disassembly during chloroplast constriction, perhaps by limiting
the reassembly of FtsZ filaments as division proceeds.
The interaction of FtsZ2 with PARC6 (Figs. 2 and 3E)
might facilitate this activity by bringing ARC3 and
FtsZ2 into close proximity (Fig. 7B). Because the PARC6
localization pattern implies that it remains associated
with the newly formed poles immediately following
separation of the daughter plastids (Glynn et al., 2009),
PARC6, by recruiting ARC3 to the midplastid, could
also (or instead) play a role in preventing the premature
assembly and misplacement of Z rings at the new poles
once division is complete (Osteryoung and Pyke, 2014).
ARC6, which also localizes to the midplastid in the
IEM (Vitha et al., 2003), has been proposed to function
partly as a membrane tether for the Z ring by virtue of
its interaction with FtsZ2 (Maple et al., 2005). Work on
bacterial FtsZ has shown that membrane tethering is an
integral part of the mechanism driving Z-ring-mediated
membrane constriction (Osawa et al., 2008). However,
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Role of PARC6 in Chloroplast Division
single-particle tracking of FtsZ filaments in an arc6
mutant, while supporting such a role for ARC6, also
suggested the presence of an additional membrane
anchor that interacts transiently with FtsZ (Johnson
et al., 2013). Our finding that FtsZ2 also interacts with
PARC6 implicates PARC6 as the alternative anchor and
suggests that Z-ring tethering by PARC6 could contribute to IEM constriction during chloroplast division
(Fig. 7). However, in arc6 mutants, where PARC6 is
presumably still present, FtsZ localizes to very small
filaments and puncta and chloroplast division is drastically impaired (McAndrew et al., 2001; Vitha et al.,
2003), whereas in parc6-1 mutants, where ARC6 is
presumably still present, FtsZ forms multiple Z rings
and spirals and chloroplasts still divide to some extent
(Fig. 5; Supplemental Fig. S2; Glynn et al., 2009). These
findings, along with the related ARC6 and PARC6
overexpression phenotypes (Vitha et al., 2003; Fig. 5A),
indicate that ARC6 likely plays a more important role
than PARC6 in Z-ring anchoring and membrane constriction. Intriguingly, the C-terminal peptide of FtsZ2,
which is conserved in bacterial FtsZ, mediates FtsZ2
interaction with both PARC6 and ARC6 (Fig. 4; Maple
et al., 2005). These results suggest the possibility of
competitive binding of FtsZ filaments by these two
proteins, which could be important in regulating Z-ring
dynamics during constriction.
Previously, we demonstrated that PARC6 acts genetically downstream of ARC6 to direct the midplastid
localization of PDV1 (Glynn et al., 2009), but the molecular basis of this process was unclear. Analysis of
PARC6 topology enabled us to identify the IMS region
of PARC6 and establish that it interacts specifically with
the IMS region of PDV1 but not PDV2. Similar to the
specific interaction between ARC6 and PDV2 (Glynn
et al., 2008), mutation of a conserved C-terminal Gly
drastically attenuated this interaction (Fig. 6). Because
the same mutation in pdv1-2 abolished PDV1 localization
to the division site in vivo (Miyagishima et al., 2006), our
results provide strong evidence that direct interaction
between PARC6 and PDV1 in the IMS positions PDV1 to
the midplastid during chloroplast division. These findings, together with our results showing that PDV1 can
recruit DRP5B from the cytosol to the OEM in a PDV2independent manner (Miyagishima et al., 2006), possibly
via direct interaction (Holtsmark et al., 2013), and that
PARC6 interacts with FtsZ2 in the stroma (Figs. 2 and 3),
strongly support a role for PARC6 in coordinating the
inner Z ring and the outer DRP5B ring across the envelope membranes during chloroplast division in Arabidopsis (Fig. 7A).
CONCLUSION
Although PARC6 presumably arose in vascular
plants by the duplication of ARC6 (Glynn et al., 2009)
and has retained an ARC6-like topology and partially
analogous role in coordination of the division machinery, PARC6 and ARC6 have evolved antagonistic
functions in regulating Z-ring dynamics. ARC6 positively regulates FtsZ assembly, probably at least in part
by tethering FtsZ to the IEM at the division site (Vitha
et al., 2003; Maple et al., 2005), while PARC6, despite its
interaction with FtsZ2, functions overall as a negative
regulator of FtsZ assembly (Glynn et al., 2009), most
likely as a consequence of its interaction with ARC3.
Furthermore, ARC6 and PARC6 have evolved unique
roles in positioning the paralogous proteins PDV2 and
PDV1, respectively, at the division site. Thus, the evolution of PARC6 and PDV1 via the duplication of ARC6
and PDV2 in vascular plants (Miyagishima et al., 2006;
Glynn et al., 2009; Okazaki et al., 2009; Osteryoung and
Pyke, 2014) has increased the complexity of the chloroplast division machinery in this group of organisms.
Future studies on the physical and functional interactions between PARC6, ARC6, and their binding partners will yield further insight into their distinct roles in
the division process.
MATERIALS AND METHODS
Plasmid Construction for Chloroplast Import, Yeast
Two-Hybrid, and Schizosaccharomyces pombe
Colocalization Experiments
All fragments of chloroplast division genes used for generating new constructs for chloroplast import, yeast two-hybrid, and S. pombe experiments were
amplified by PCR from complementary DNA sequences. Primers are shown in
Supplemental Table S1. Details of plasmid construction are described in the
relevant sections below. For constructs generated using Gibson assembly reactions (Gibson et al., 2009), specified below, PCR products were used directly.
Except where indicated, other PCR products were digested with the restriction
enzymes shown in Supplemental Table S1 prior to insertion into the relevant
vectors. All constructs were sequenced prior to use in experiments.
In Vitro Translation of Precursor Protein
The entire coding sequence of Arabidopsis (Arabidopsis thaliana) PARC6 was
amplified with primer set MZ1151/MZ1077 (Supplemental Table S1). The
resulting product was cloned into pBluescript SK+ and was subsequently mutagenized using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen)
using primer sets PARC6-DTMD1-F1/PARC6-DTMD1-R1 and PARC6-DTMD2F2/PARC6-DTMD2-R2 according to the manufacturer’s protocol to generate the
constructs PARC6(DTMD1) and PARC6(DTMD2), respectively. PARC6(DTMD1)
was used as a template with primer set PARC6-DTMD2-F2/PARC6-DTMD2-R2
to create PARC6(DTMD1/2). The SSU and ARC6 constructs used as controls have
been described previously (Olsen and Keegstra, 1992; Vitha et al., 2003).
Radiolabeled precursor proteins were generated by in vitro translation in the
presence of [35S]L-Met (Perkin-Elmer; catalog no. NEG009T005MC) using the
TNT Coupled Reticulocyte Lysate System according to the manufacturer’s
protocol (Promega). After translation, the labeled proteins were diluted with
an equal volume of nonradioactive 50 mM L-Met in import buffer (50 mM
HEPES-KOH, pH 8, and 330 mM sorbitol).
Isolation of Pea Chloroplasts
Intact chloroplasts were isolated from 8- to 12-d-old pea (Pisum sativum ‘Little
Marvel’, dwarf variety; Livingston Seed) seedlings and purified over a Percoll
gradient as described previously (Froehlich, 2011). Intact pea chloroplasts were
reisolated and resuspended in import buffer at a concentration of 1 mg chlorophyll mL21.
Chloroplast Import and Protease Protection Assays
Large-scale import and protease protection assays were performed as described (Froehlich, 2011). Reactions containing 150 mL of chloroplasts (1 mg
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Zhang et al.
chlorophyll mL21) prepared as above, 4 mM Mg-ATP, and 10% (v/v) of the
radiolabeled precursor protein in a final volume of 450 mL of import buffer were
incubated for 30 min at room temperature under room light. Reactions were
then divided into three 150-mL aliquots. One portion was incubated for 30 min
on ice. The other portions were incubated for 30 min on ice with either thermolysin or trypsin. Thermolysin and trypsin treatments were quenched with
50 mM EDTA or trypsin inhibitor, respectively. Chloroplasts were then recovered by centrifugation through a 40% (v/v) Percoll cushion. Recovered chloroplasts were lysed by the addition of lysis buffer (25 mM HEPES, pH 8, and
4 mM MgCl2) and then fractionated into total soluble and total membrane
fractions. All fractions were subsequently analyzed using SDS-PAGE. After
electrophoresis, gels were subjected to fluorography and exposed to x-ray film
(Eastman Kodak).
platform. After extensive washing, samples were eluted from beads into 200 mL
of elution buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 10 mM
maltose). To prevent protein degradation, an EDTA-free protease inhibitor
cocktail (Roche Molecular Biochemicals) was included in the elution buffer at a
concentration of one tablet per 50 mL of buffer.
Approximately 12.5% of the eluted material was applied in each lane for
separation on 15% SDS-PAGE gels. Separated proteins were transferred to a
polyvinylidene difluoride membrane (EMD Millipore) and probed with antiMBP (New England Biolabs) or anti-GST (Sigma-Aldrich) antibodies at dilutions
of 1:20,000 and 1:10,000, respectively. Blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody, and immunoreactive bands were detected by chemiluminescence (Thermo Fisher
Scientific).
Yeast Two-Hybrid Assays
Heterologous Expression of Fluorescent Fusion Proteins in
S. pombe
The PARC677-573 and PARC6357-573 fragments were amplified using primer
sets MZ-C001/MZ-C002 and MZ-C004/MZ-C002, respectively, and inserted
into the pGAD-T7 vector (Clontech) between the NdeI and BamHI sites. The
ARC3MORN construct was generated by amplifying the region encompassing
amino acids 598 to 741 of ARC3 using the primer set 598F/ARC3-2 and
inserting the product into the pGBK-T7 vector (Clontech). The ARC3DMORN
construct (previously called ARC3-MORN) is described by Glynn et al. (2009). The
PARC6597-819 fragment was amplified with the primer set JG-MiaF/JG-MiaR
and inserted into pGAD-T7 between the NdeI and XmaI sites to create the
PARC6IMS construct. PDV1IMS(G272D) was generated by PCR-based mutagenesis
of PDV1IMS (Glynn et al., 2008) using primer set JG-294/MZ1132 and cloned
into pGBK-T7 between the NdeI and XhoI sites. All other yeast two-hybrid
constructs were reported previously (Glynn et al., 2008, Schmitz et al., 2009).
Yeast cells were grown on plates containing synthetic dropout medium either
with (SD/-Trp-Leu) or without (SD/-Trp-Leu-His) His (Clontech). Assays
shown in Figures 2 and 4A were performed in the yeast strain Y2HGold
(Clontech). Positive interactions in this strain were detected by activation of
both the HIS3 reporter, as indicated by growth in SD/-Trp-Leu-His, and the
AUR1-C reporter, as indicated by growth on 0.1 mg mL21 aureobasidin A
(Clontech) included in the medium. HIS3 reporter assays shown in Figure 6
were performed in the yeast stain AH109 (Clontech) in the presence of 2.5 mM
3-amino-1,2,4-triazole (Sigma-Aldrich). Cell growth and transformation were
performed according to the manufacturer’s protocol (Clontech). Interactions
were tested in at least three independent assays, and consistent results were
obtained.
Pull-Down Assays
A fragment of PDV1 encoding amino acids 226 to 272 was PCR amplified
from complementary DNA using primer set JG-498 and JG-499, digested with
BamHI and NotI, and then inserted into pGEX-4T-2 (GE Healthcare Life Sciences) to create the construct GST-PDV1IMS. To make GST-PDV1IMS(G272D),
PDV1IMS(G272D) was generated by PCR-based mutagenesis using primers JG-498
and MZ-1132 and then cloned into pGEX-4T-2. A fragment encoding amino
acids 597 to 819 of PARC6 was amplified with primers BA01 and BA02, digested
by EcoRI and HindIII, and then cloned into pMAL-c4x (New England Biolabs),
yielding MBP-PARC6IMS. All three constructs and the empty vectors pGEX-4T-2
encoding GST and pMAL-c4x encoding MBP were transformed into BL21
(DE3) Codon Plus cells (Stratagene). Cells harboring pGEX-4T-2, GST-PDV1IMS,
or GST-PDV1IMS(G272D) were cultured in normal Luria-Bertani medium (10 g of
NaCl, 10 g of tryptone, and 5 g of yeast extract per L), while cells harboring
MBP-PARC6IMS were grown in low-salt Luria-Bertani medium (5 g of NaCl,
10 g of tryptone, and 5 g of yeast extract) with 0.2% (w/v) Glc. Expression of all
constructs was induced with 0.5 mM isopropylthio-b-galactoside and cultured
at 37°C for 3 h. Cell pellets were resuspended in lysis buffer (20 mM Tris-HCl,
pH 7.4, 200 mM NaCl, and 1 mM EDTA) with EDTA-free protease inhibitor
cocktail (Roche Molecular Biochemicals) and then lysed by sonication. All recombinant proteins in crude cell extracts were roughly quantified by comparison with 2 mg of bovine serum albumin protein on SDS-PAGE gels stained with
Coomassie Blue. For pull-down assays, the lysis buffer only or approximately
700 mL of clarified cell lysate containing 10 mg of GST, GST-PDV1IMS, or GSTPDV1IMS(G272D) was incubated at 4°C for 4 h in 2-mL microtubes with 50 mL of
equilibrated glutathione-Sepharose 4B beads (GE Healthcare Life Sciences)
preequilibrated with lysis buffer. The beads in each tube were then washed
extensively with lysis buffer and incubated with 500 mL of cell lysate containing
approximately 10 mg of MBP-PARC6IMS at 4°C for another 4 h on a rocking
The expression vectors pREP41X and pREP42X were used for all experiments
in S. pombe (Basi et al., 1993; Forsburg, 1993). Sequences encoding the PARC6
stromal region (PARC677-573) and mCherry (Shaner et al., 2004) were amplified
using the primer sets CC-4/CC-8 and CC-6/CC-7, respectively, and cloned into
pREP41X and pREP42X digested with XhoI and BamHI using the Gibson assembly method, yielding pREP41X-PARC677-573-mCherry and pREP42XPARC677-573-mCherry. To obtain pREP41X-mCherry, the mCherry fragment was
PCR amplified using primer set CC-10/CC-7 and cloned into pREP41X
digested with XhoI and BamHI. The pREP41X-FtsZ1-eYFP and pREP42X-FtsZ2eCFP constructs and cell growth and transformation conditions were described
previously (TerBush and Osteryoung, 2012). To obtain pREP42X-FtsZ249-478mCerulean, FtsZ249-478 and mCerulean (Shaner et al., 2004; Papapetrou et al., 2009;
Addgene) fragments were PCR amplified using primer sets AT264/AT299 and
AT7/AT297, respectively, and cloned into BamHI-digested pREP42X by Gibson
assembly. pREP42X-FtsZ249-457-mCerulean was generated similarly using primer
sets AT264/AT288 and AT7/AT297, respectively. An FtsZ2F466A-mCerulean fragment was PCR amplified from the pREP42X-FtsZ249-478-mCerulean construct using
primer sets AT264/AT350 and AT349/AT297 and cloned into BamHI-digested
pREP42X by Gibson assembly to generate pREP42X-FtsZ2F466A-mCerulean. To
make pREP41X-ARC668-614-mVenus, ARC668-614 and mVenus (A206K mutation of
Venus; D.A. Moore and H.P. Erickson, personal communication; Nagai et al.,
2002; Zacharias et al., 2002) were PCR amplified using primer sets AT168/AT139
and AT95/AT147, respectively, and cloned into pREP41X digested with XhoI and
BamHI by Gibson assembly.
S. pombe cells expressing chloroplast division fluorescent fusion proteins
were imaged by differential interference contrast and epifluorescence microscopy with a Leica DMRA2 microscope equipped with an HCX PL FLUOTAR
1003 (1.30 numerical aperture) oil-immersion objective (Leica) and a CCD
camera (Retiga Exi; QImaging). Two microliters of liquid cell culture was
pipetted onto a polylysine-coated slide and covered with a No. 1.5 coverslip. All
images were collected at room temperature. Z stacks were taken at 0.5-mm
increments, and nearest neighbor deconvolution with 70% haze removal was
performed with Image-Pro Plus 7.0 software (Media Cybernetics) on Z planes
with in-focus fluorescence signal. Further image manipulations were performed with Fiji (ImageJ) software (http://fiji.sc/Fiji). Projections were made
from Z stacks using the max intensity algorithm and were falsely colored as
indicated in the figure legends. Colocalization of PARC677-573-mCherry with
FtsZ1-eYFP, FtsZ2-eCFP, FtsZ249-478-mCerulean, FtsZ249-457-mCerulean, or
FtsZ2F466A-mCerulean and mCherry with FtsZ2-eCFP was quantified by creating a composite image of the two coexpressed fluorescent signals from a
deconvoluted Z stack, cropping the image to contain only the cell being analyzed, unmerging the two channels, using the Coloc2 plugin within Fiji to
calculate an individual PCC value, and then averaging all PCC values for each
coexpression strain 6 SE.
Generation of Transgenic Plants
Gibson assembly (Gibson et al., 2009) was used to generate constructs for
expression in transgenic plants. The bacterial artificial chromosome genomic
clone MVI11 (Arabidopsis Biological Resource Center) was used as a template
with primers CC-27 and CC-28 (Supplemental Table S1) to produce a PARC6Myc fragment by PCR. The PARC6-Myc fragment was cloned into the
pCAMBIA1300-20 vector (Zhang et al., 2013) digested with BamHI and SacI to
produce 35S::PARC6-Myc. For complementation of parc6-1, an approximately
1.5-kb genomic fragment upstream of the PARC6 start codon (Zhang et al.,
260
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Role of PARC6 in Chloroplast Division
2009) was amplified using primers CC-31 and CC-28. The resulting PCR product and the PARC6-Myc fragment were cloned into pCAMBIA1300-20
digested with PstI and SacI to produce PARC6::PARC6-Myc. Constructs were
introduced into Agrobacterium tumefaciens GV3101 by electroporation, and
plants were transformed by floral dipping (Zhang et al., 2006). T1 transgenic
plants were selected on plates containing 20 mg mL21 hygromycin, then
transferred to soil and grown as described (Kadirjan-Kalbach et al., 2012).
Protein Extraction and Immunoblot Analysis
For 35S::PARC6-Myc overexpression lines, 9-d-old T2 transgenic seedlings
were selected on hygromycin as above and whole seedlings were harvested
directly from plates. For PARC6::PARC6-Myc complementation lines, leaf tissue
was harvested from 4-week-old T1 transgenic plants. Protein samples were
prepared as described previously (Kadirjan-Kalbach et al., 2012) with slight
modification. Briefly, tissue was ground to powder in liquid nitrogen using an
MP FastPrep-24 Tissue and Cell Homogenizer (MP Biomedicals). The powder
was suspended in 43 SDS-PAGE loading buffer (40% glycerol, 240 mM TrisHCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, and 5% b-mercaptoethanol) at a
concentration of 0.5 mg fresh weight mL21. Proteins were separated on 10%
SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking, the
membrane was incubated overnight with anti-PARC6 (1:500 dilution; Glynn
et al., 2009), anti-FtsZ1 (1:20,000 dilution), or anti-FtsZ2-1 (1:14,000 dilution;
Stokes et al., 2000) antibodies. Following washing, blots were incubated with
horseradish peroxidase-conjugated goat anti-rabbit secondary antibody
(1:5,000 dilution) for 1 h at room temperature. The bands were detected using
SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
Analysis of Chloroplast Phenotypes
Samples were collected from expanded rosette leaves of 3- to 4-week-old
plants. Leaf tips were fixed in 3.5% glutaraldehyde for 3 h followed by heating in
0.1 M Na2EDTA (pH 9) at 50°C for an additional 1.5 h (Pyke and Leech, 1991).
Leaf samples were viewed with a Leica DMI3000B inverted microscope
equipped with a Leica DFC320 camera.
Immunofluorescence Staining
Immunofluorescence staining of leaf samples obtained from 3- to 4-week-old
plants was performed essentially as described (Yoder et al., 2007; Vitha and
Osteryoung, 2011). Five-micrometer-thick sections were incubated with
affinity-purified anti-FtsZ1 (1:3,500 dilution) or anti-FtsZ2-1 (1:3,500 dilution)
antibodies (Yoder et al., 2007). Alexa Fluor 488-conjugated goat anti-rabbit
secondary antibody (Invitrogen) was used at a dilution of 1:500. Samples
were observed with a Leica DMRA2 microscope as described above using
Leica filters L5 (480 nm excitation/505 nm emission) and TX2 (560 nm
excitation/595 nm emission) to visualize Alexa Fluor 488 and chlorophyll
fluorescence, respectively.
Sequence data from this article can be found in The Arabidopsis Information
Resource or GenBank/EMBL databases under the following names and accession numbers: PARC6 (AT3G19180), ARC6 (AT5G42480), ARC3 (AT1G75010),
FtsZ1 (AT5G55280), FtsZ2-1 (AT2G36250), PDV1 (AT5G53280), and PDV2
(AT2G16070). The mutant used in this study is parc6-1 (SALK_100009).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Additional examples of PARC677-573-mCherry
coexpressed with the various forms of FtsZ2-mCerulean.
Supplemental Figure S2. Complementation of parc6-1 mutants with the
PARC6-Myc fusion gene.
Supplemental Table S1. Primers used in this study.
ACKNOWLEDGMENTS
We thank Jonathan Glynn, Aaron Schmitz, and Bradley Abramson for
providing yeast two-hybrid constructs, Katie Porter for generating the
FtsZ2 F466A mutation construct for use in S. pombe, Larissa Osterbaan and
Geoffry Davis for conducting preliminary PARC6-PDV1 interaction assays,
Maren Friesen for providing a plasmid encoding mCherry, Desmond Moore
and Harold P. Erickson for providing the mVenus construct, and Chen Wang
for assistance with the preparation of Figure 7.
Received September 15, 2015; accepted November 2, 2015; published November 2, 2015.
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