T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 179, No. 6, December 17, 2007 1123–1131
To understand the mechanism of membrane fi ssion, we identi-
fi ed and used a compound called Ilimaquinone (IQ), which ve-
siculates the Golgi apparatus via a trimeric G protein subunit βγ
and a serine/threonine kinase called protein kinase D (PKD)–
dependent process (Takizawa et al., 1993; Jamora et al., 1997,
1999). Importantly, PKD is necessary for the biogenesis of TGN
to cell surface transport carriers (Liljedahl et al., 2001; Bard and
Malhotra, 2006). The binding of PKD to TGN requires DAG
(Baron and Malhotra, 2002) and is activated by Golgi-associated
PKCη (Diaz Anel and Malhotra, 2005). PKD activates the lipid
kinase activity of PI4kinase IIIß to generate phosphoinositide
4-phosphate (PI4P) from PI, and regulates the binding of
ceramide transfer protein CERT to PI4P. PI4P is required for TGN-
to-cell surface transport (Walch-Solimena and Novick, 1999;
Audhya et al., 2000; Godi et al., 2004; Hausser et al., 2005,
2006; Fugmann et al., 2007). The evidence for PKD’s role in
the formation of TGN to cell surface transport carriers is though
use of a kinase-dead (KD) form and pharmacological inhibitors.
The best evidence for PKD’s direct involvement in membrane
fi ssion requires that its depletion inhibits protein secretion.
However, the problem is exacerbated by the fact that there are
three isoforms of PKD in the mammalian cells (1, 2, and 3)
(Rykx et al., 2003), and all are involved in the formation of
basolaterally directed transport carriers (Yeaman et al., 2004).
We believe we have now addressed this issue. Our fi ndings
reveal that HeLa cells contain predominantly PKD2 and PKD3,
and virtually no PKD1. PKD2 and PKD3 dimerize at the TGN
and we suggest they activate different substrates. Importantly,
depletion of PKD2 and PKD3 by siRNA inhibits TGN-to-cell
surface transport. Under these conditions, cargo containing
tubules and reticular membranes accumulate at the TGN. In con-
trast, overexpression of an activated PKD causes extensive vesi-
culation of TGN. These results demonstrate convincingly that
PKD is a bona fi de component of membrane fi ssion used to
regulate the number and size of TGN-to-cell surface transport
carriers depending on the physiological (cargo) needs.
Results and discussion
Depletion of PKD2 and PKD3 inhibits TGN-
to-cell surface protein transport
RT-PCR–based analysis revealed that of the three PKD iso-
forms, only PKD2 and PKD3 were expressed in HeLa cells
(Fig. 1 A). These results were confi rmed by quantitative RT-
PCR (qRT-PCR): PKD1-specifi c mRNA is virtually undetect-
able (10- and 12-fold lower) compared with PKD2 and PKD3,
respectively (Fig. 1 B). Specifi c siRNAs were designed to de-
plete PKD2 and PKD3 in HeLa cells. Western blotting with
specifi c antibodies revealed a 70–75% reduction in the level
of PKD2 and PKD3, respectively (Fig. 1, C and E). By com-
parison, the level of β-actin was not affected by PKD-specifi c
siRNAs (Fig. 1 D).
To test the effect of PKD2 and PKD3 depletion on protein
secretion, control cells and depleted HeLa cells were cotransfected
with a plasmid expressing HRP containing the N-terminal sig-
nal sequence (SS) as described previously (Bard et al., 2006)
Dimeric PKD regulates membrane fi ssion to form
transport carriers at the TGN
Carine Bossard,1 Damien Bresson,2 Roman S. Polishchuk,3 and Vivek Malhotra1
1Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093
2La Jolla Institute for Allergy and Immunology, Developmental Immunology 3, La Jolla, CA 92037
3Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH) 66030, Italy
TGN to cell surface transport carriers. We now provide
defi nitive evidence that PKD has a function in membrane
fi ssion. PKD depletion by siRNA inhibits traffi cking from
rotein kinase D (PKD) is recruited to the trans-Golgi
network (TGN) through interaction with diacylglyc-
erol (DAG) and is required for the biogenesis of
the TGN, whereas expression of a constitutively active PKD
converts TGN into small vesicles. These fi ndings demon-
strate that PKD regulates membrane fi ssion and this activ-
ity is used to control the size of transport carriers, and
to prevent uncontrolled vesiculation of TGN during pro-
Correspondence to Vivek Malhotra: email@example.com
V. Malhotra’s present address is Cell and Developmental Biology program, Centre
de Regulació Genòmica (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.
Abbreviations used in this paper: CA, constitutively active; KD, kinase dead;
PKD, protein kinase D; PLAP, placental alkaline phosphatase; SS, signal sequence;
JCB • VOLUME 179 • NUMBER 6 • 2007 1124
together with a plasmid expressing placental alkaline phospha-
tase (PLAP), a GPI-anchored protein that contains an apical
sorting signal (Lisanti et al., 1990; Lipardi et al., 2000). The ac-
tivities of HRP and PLAP released into the medium were mea-
sured by chemiluminescence (Bard et al., 2006). HRP secretion
is inhibited by 82% in cells transfected by PKD2 siRNA and
by 80% in cells transfected by PKD3 siRNA compared with
control siRNA-transfected cells (Fig. 2 A). None of the PKD
siRNAs have any effect on PLAP secretion (Fig. 2 B). These
fi ndings strengthen our previous proposal that similarly to po-
larized cells that have distinct apical and basolateral targeting
pathways, nonpolarized HeLa cells sort proteins into apical-
like (PKD-independent) and basolateral-like (PKD-dependent)
pathways (Yeaman et al., 2004). Simultaneous depletion of
PKD2 and PKD3 from HeLa cells did not have a synergistic
effect in inhibiting HRP secretion (unpublished data). Because
siRNA-based depletion is not complete, we suggest the residual
PKD is suffi cient to support traffi cking from the TGN to the
cell surface. To further ascertain the involvement of PKD2 and
PKD3 in traffi cking, their involvement in the transport of vesic-
ular stomatitis virus (VSV)–G protein was monitored. HeLa
cells were fi rst cotransfected with either siRNA specifi c for
PKD2, PKD3, or a control, and a fl uorescent-labeled siRNA to
identify transfected cells. After 30 h, the cells were transfected
with the tsO45 strain of VSV-G–GFP, which has a thermosensi-
tive mutation causing it to unfold in the ER at the nonpermissive
temperature of 39.5°C. Upon shifting cells to the permissive
temperature of 32°C, tsO45VSV-G protein folds and is trans-
ported to the cell surface. The amount of VSV-G at the cell
surface was quantifi ed by immunofl uorescence fl ow cytometry
permitting analysis of more than 5,000 cells transfected with
both siRNA and VSV-G-GFP. In control cells, VSV-G protein
was found at the cell surface within 40 min of shift to 32°C.
In PKD2- and PKD3-depleted cells (either individually or to-
gether), 50% less VSV-G was found at the cell surface (Fig. 2 C).
This is a signifi cant effect on the traffi cking of VSV-G to the cell
surface considering that siRNA-based depletion of PKD is not
complete, and the residual levels (25–30%) would support traf-
fi cking, albeit at a slower rate.
Immunoelectron microscopy was used to monitor effects
on the secretion of ss-HRP (cargo) in cells depleted of PKD2
and PKD3. Compared with control cells (Fig. 2 D), PKD2 and
PKD3 depletion resulted in accumulation of HRP containing
highly fenestrated membranes, and large tubules in the trans
region of the Golgi stacks (Fig. 2, E and F). To quantitate the
number of tubules in the TGN, 20 different Golgi stacks were
visualized. In control cells there were 1 to 3 tubules per stack,
whereas in PKD-depleted cells the number of tubules increased
to 3 to 6 per stack. In sum, PKD depletion results in accumula-
tion of HRP in the TGN and on average a 2.9-fold increase in
the number of HRP-containing tubules per Golgi stack. We suggest
that these tubules and fenestrated HRP-containing membranes
would ordinarily be converted into small transport carriers by
membrane fi ssion. However, depletion of PKD2 and PKD3 in-
hibits events leading to membrane fi ssion, thus accumulating
cargo (in this instance HRP) in tubular elements.
PKD2 and PKD3 dimerize in vitro and
in vivo and transphosphorylate
Why does depletion of either PKD2 or PKD3 affect Golgi-
to-cell surface transport? Why is the other (nondepleted) isoform
not functional under such conditions? Do these forms dimerize
to activate downstream targets? To test this hypothesis, PKD2
and PKD3 were immunoprecipitated separately from HeLa cell
lysates and Western blotted with anti-PKD3 and anti-PKD2,
respectively. Western blotting pure recombinant PKD2 and 3
confi rmed the specifi city of the antibodies to PKD2 and PKD3
(Fig. 3 A). The endogenous PKD2 and PKD3 were found to co-
precipitate (Fig. 3 B). Exogenously expressed GST-PKD2 and
Flag-PKD3 also coprecipitate specifi cally (Fig. 3 C). GST alone
Figure 1. Relative expression of PKD iso-
forms in HeLa cells and their depletion by siRNA.
(A) Analysis of mRNA expression by RT-PCR
shows that PKD2 and PKD3 are the only PKD
isoforms expressed in HeLa cells. RT-PCR reaction
without the reverse transcriptase (RT−) was used
as a negative control and PCR with the corre-
sponding PKD cDNA (C) as a positive control.
(B) Quantitative real-time RT-PCR analysis was
performed on RNA extracted from HeLa cells.
Bars represent the mean (±SD) of the relative
mRNA expression of each PKD isoform com-
pared with the average expression of β-actin.
*, P < 0.01 compared with PKD1. (C) PKD2 and
PKD3 protein levels in HeLa cells transfected
with the indicated siRNA were detected by
immunoblot analysis after immunoprecipitation
from 100 μg of cell lysate using anti-PKD2 and
anti-PKD3 antibodies, respectively. (D) β-Actin
expression in the lysates used for immunopreci-
pitation was monitored as a loading control.
(E) The effect of PKD2 and PKD3 siRNA was
quantifi ed by densitometry and normalized to
the expression of PKD2 and PKD3, respectively,
in cells transfected with control siRNA.
MEMBRANE FISSION BY PKD • BOSSARD ET AL.1131
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