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of June 17, 2017.
Mechanisms Regulating the Positioning of
Mouse p47 Resistance GTPases LRG-47 and
IIGP1 on Cellular Membranes: Retargeting
to Plasma Membrane Induced by
Phagocytosis
Sascha Martens, Katja Sabel, Rita Lange, Revathy Uthaiah,
Eva Wolf and Jonathan C. Howard
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2004; 173:2594-2606; ;
doi: 10.4049/jimmunol.173.4.2594
http://www.jimmunol.org/content/173/4/2594
The Journal of Immunology
Mechanisms Regulating the Positioning of Mouse p47
Resistance GTPases LRG-47 and IIGP1 on Cellular
Membranes: Retargeting to Plasma Membrane Induced
by Phagocytosis1
Sascha Martens,2* Katja Sabel,* Rita Lange,* Revathy Uthaiah,3* Eva Wolf,† and
Jonathan C. Howard2*
T
he p47 GTPases are some of the most potent, known resistance factors against intracellular pathogens in the mouse
(1–3). Mice with targeted deletions of single members of the
p47 GTPases showed complete loss of immune control of certain
bacterial and protozoan infections. In particular, LRG-47 has been
shown to be essential for resistance against Toxoplasma gondii and
Listeria monocytogenes (2), and most recently against Mycobacterium tuberculosis (4) and Mycobacterium avium (5). LRG-47 has
been copurified with phagosomes isolated from Mycobacterium-infected macrophages and was shown to promote acidification and thus
maturation of the phagosome (4). However, the cellular localization
and mode of recruitment of LRG-47 to the phagosome and the nature
of its association with cellular membranes are unknown.
All p47 GTPases tested so far, including LRG-47, are abundantly induced by IFN-␥ from low resting levels in vivo and in
vitro (6) and have been shown to confer cell-autonomous immunity against intracellular pathogens (1, 2, 4, 7, 8). So far 6 family
*Institute for Genetics, University of Cologne, Cologne, Germany; and †Department
of Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany
Received for publication February 6, 2004. Accepted for publication May 18, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Financial support for this project was provided by the Sonderforschungsbereiche
SFB243 and SFB635, and the Schwerpunktprogramm SP1110 of the Deutsche Forschungsgemeinschaft (DFG), Centre for Molecular Medicine of the Medical Faculty,
University of Cologne, and the Land Nordrhein-Westfalen through the University of
Cologne. R.U. acknowledges a stipendium from the DFG Graduate College “Genetik
zellulärer Systeme” at the University of Cologne.
2
Address correspondence and reprint requests to Drs. Sascha Martens or Jonathan
C. Howard, Institute for Genetics, University of Cologne, Zülpicher Strasse 47,
50674 Cologne, Germany. E-mail addresses: [email protected] and
[email protected]
3
Current address: Department of Cellular Biochemistry and Biophysics, Memorial
Sloan-Kettering Cancer Center, New York, NY 10021.
Copyright © 2004 by The American Association of Immunologists, Inc.
members, namely TGTP (9), IRG-47 (10), IIGP1 (6), GTPI (6),
IGTP (11), and LRG-47 (12), have been described in the mouse,
although the gene family in the C57BL/6 mouse has 23 members
(C. Bekpen, J. Hunn, L. Guethlein, D. M. Dunn, E. Glowalla, I.
Parvanova, and J. C. Howard, manuscript in preparation). The 6
published p47 GTPases fall into two sequence subgroups (6), GKS
and GMS, defined by variation in the otherwise universally conserved G1 motif. This is the familiar GX4GKS in IIGP1, TGTP,
and IRG-47, but GX4GMS in LRG-47, IGTP, and GTPI. Members
of both subgroups are involved in cell-autonomous resistance and
the function of at least one member, IGTP, is required in cells of
both hemopoietic and nonhemopoietic origin (13). This is consistent with the finding that the p47 GTPases are induced by IFN-␥ in
all cell types tested so far (13).
Recently, the crystal structure of IIGP1 has been determined
(33). The Ras-like G domain is preceded by a three-helix bundle
carrying an N-terminal myristoylation motif, and is followed by a
larger C-terminal domain that is also predominantly helical. Both
N- and C-terminal regions of the different p47 GTPases are highly
variable in sequence (6). However, colinear stretches of high
homology occur throughout the molecule, and largely shared
secondary structure predictions strongly suggest that all the p47
GTPases share a fundamentally similar structure (33).
Three family members, namely IGTP, IIGP1, and LRG-47, have
been shown to be associated with internal membrane compartments. IGTP was found to be endoplasmic reticulum (ER)4 associated in mammary gland-derived C127 cells in a GTP-independent
manner (14); IIGP1 has been reported to localize predominantly to
4
Abbreviations used in this paper: ER, endoplasmic reticulum; LAMP1, lysosomeassociated membrane protein 1; 3D, three-dimensional; EGFP, enhanced GFP.
0022-1767/04/$02.00
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The recently identified p47 GTPases are one of the most effective cell-autonomous resistance systems known against intracellular
pathogens in the mouse. One member of the family, LRG-47, has been shown to be essential for immune control in vivo of Listeria
monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Mycobacterium avium, possibly by promoting acidification of
the phagosome. However, the intracellular localization of LRG-47, and the nature of its association with the phagosomal or any
other membrane system is unknown. In this study, we show that LRG-47 is a Golgi-associated protein in the IFN-stimulated cell,
which is rapidly recruited to active plasma membrane upon phagocytosis and remains associated with phagosomes as they mature.
We show that the Golgi localization of LRG-47 is dependent on the integrity of an amphipathic helix near the C terminus, whereas
the plasma membrane localization depends on an unidentified signal associated with the G domain. Unlike LRG-47, but like the
published p47 resistance GTPase, IGTP, a further p47 GTPase, IIGP1, is associated with the endoplasmic reticulum. However,
unlike IGTP, IIGP1 is associated with the endoplasmic reticulum by an N-terminal myristoylation modification. Thus, the p47
GTPases are a diverse battery of intracellular defense factors dynamically associated with different membrane systems. The
Journal of Immunology, 2004, 173: 2594 –2606.
The Journal of Immunology
Materials and Methods
Cells and tissue culture
RAW 264.7, L929 mouse fibroblasts, and TIB-75 hepatocytes (16) were
cultured in IMDM supplemented with 10% FCS (Sigma-Aldrich, Deisenhofen, Germany), 2 mM L-glutamine (Invitrogen Life Technologies, Eggenstein, Germany), 1 mM sodium pyruvate (ICN, Eschwege, Germany),
100 U/ml penicillin (Invitrogen Life Technologies), and 100 ␮g/ml streptomycin (Invitrogen Life Technologies). Cells were induced with mouse
IFN-␥ (Cell Concepts, Umkirch, Germany) at a concentration of 200 U/ml
for the time spans indicated in the figures. Peritoneal macrophages were
isolated from the peritoneal cavity of CB20 mice and cultured in six-well
plates with coverslips. Cells were grown in IMDM and washed several
times to remove nonadherent cells over a period of 36 – 48 h before
experiments.
Antisera and Abs
Polyclonal anti-IIGP1 antiserum (165) was raised against the full-length
protein. The purification of IIGP1 as a cleaved GST-fusion protein is described elsewhere (17). Rabbits were immunized against the full-length
IIGP1 protein.
Serological agents used were mouse monoclonal anti-IGTP I68120 (BD
Transduction Laboratories, Lexington, KY), mouse monoclonal anti-IIGP1
10D7 and 10E7 Abs (gift from J. Zerrahn, Max-Planck Institute, Berlin,
Germany), anti-TGTP A20 sc-11079 goat anti-peptide antiserum (Santa
Cruz Biotechnology, Santa Cruz, CA), and LRG-47 A19 sc-11075 goat
anti-peptide antiserum (Santa Cruz Biotechnology). Anti-IRG-47 2078 rabbit anti-peptide antiserum was raised against internal peptides, CKTPYQHPKYPKVIF and CDAKHLLRKIETVNVA, using the double X program
(Eurogentec, Seraing, Belgium), affinity-purified anti-rab6 rabbit antiserum
(gift from B. Goud, Centre National de la Recherche Scientifique, Paris,
France), monoclonal anti-Giantin Ab (gift from H.-P. Hauri, Biozentrum,
Basel, Switzerland (18)), anti-ERP60 rabbit antiserum (gift from T. Wileman, BBSRC, Pirbright, U.K.), monoclonal rat anti-LAMP-1 1D4B (University of Iowa, Iowa City, IA), monoclonal anti-TAP 294 (19), monoclonal anti-FLAG M2 (Sigma-Aldrich), anti-calnexin SPA-865 rabbit
antiserum (StressGen Biotechnologies, Victoria, BC, Canada), anticaveolin-1 N20 sc-894 rabbit antiserum (Santa Cruz Biotechnology), goat
anti-mouse Alexa 546/488, goat anti-rabbit Alexa 546/488, donkey antigoat Alexa 546/488, donkey anti-mouse Alexa 488, donkey anti-rabbit Alexa 488, donkey anti-rat Alexa 488 (Molecular Probes, Eugene, OR), goat
anit-mouse HRP (Amersham Biosciences, Piscataway, NJ), donkey antirabbit HRP (Amersham Biosciences), and donkey anti-goat HRP (Santa
Cruz Biotechnology).
[phospho-L-serine]; Avanti, Alabaster, AL). Lipids dissolved in chloroform
were dried under nitrogen and additionally in a rotatory evaporator to remove residual chloroform. The resulting lipid cake, hydrated by addition of
HEPES-buffered saline, was left undisturbed for 1 h at room temperature
and finally vortexed to resuspend the lipids. The lipid suspension was extruded 21 times through polycarbonate membranes with a pore size of 100
nm (Avestin, Ottawa, ON, Canada). For cosedimentation assays, 0.25 mM
phospatidylserine lipids and 0.25 ␮M IIGP1 in HEPES-buffered saline
were incubated for 30 min at 37°C followed by centrifugation for 30 min
with 125,000 ⫻ g at 4°C. The samples were further processed as described
for the cellular membranes.
Bead uptake experiments
A volume of 500 ␮l of 2-␮m carboxylated latex beads (Polysciences, Warrington, PA) were coated with collagen as follows. Beads were washed two
times with 0.1 M NaHCO3 (pH 9.6), followed by three washes with 0.02
M phosphate buffer (pH 4.5). The beads were resuspended in 675 ␮l of
phosphate buffer (pH 4.5), and 675 ␮l of 2% carbodiimide (Sigma-Aldrich)
in phosphate buffer (pH 4.5) was added. After an incubation of 4 h on a
rotator at room temperature, the beads were washed three times with phosphate buffer (pH 4.5), resuspended in 1.2 ml of borate buffer (pH 8.5)
containing 500 ␮g of collagen (type I from rat tail; Sigma-Aldrich), and
incubated overnight at room temperature on a rotator. A volume of 50 ␮l
of 0.25 M ethanolamine was added, and the beads were incubated for
another 30 min. The beads were pelleted and resuspended in borate buffer
(pH 8.5) and 10 mg/ml BSA (fraction V; Roth, Karlsruhe, Germany), and
incubated for 30 min. The beads were washed with borate buffer (pH 8.5)
and 10 mg/ml BSA, and stored in 500 ␮l of 0.02 M phosphate buffer (pH
7.4), 0.15 M NaCl, 1% BSA, 5% glycerol, and 0.1% NaN3.
L929 cells were grown on coverslips and induced with IFN-␥. Twenty
hours after induction, the collagen-coated beads were added, and the cells
were incubated for an additional 4 h. Finally, the cells were fixed with 3%
paraformaldehyde for 20 min at room temperature. A total of 3 ⫻ 106
RAW 264.7 cells was induced with 200 U/ml IFN-␥ for 48 h and incubated
with 2-␮m latex beads not coated with collagen diluted 1/1000 in IMDM
for 30 min to 4 h.
Immunofluorescence analysis
Cells were washed with PBS and fixed in 3% paraformaldehyde for 20 min
at room temperature. Cells were permeabilized with 0.1% saponin and
blocked with 3% BSA (Roth). The cells were analyzed using a Zeiss
(Oberkochen, Germany) Axioplan II fluorescence microscope equipped
with a cooled charge-coupled device camera (Quantix; Photometrix, Tucson, AZ). Image processing and two-dimensional deconvolution was done
with the MetaMorph software (version 4.5r3; Universal Imaging, Downington, PA). For three-dimensional (3D) deconvolution, the Auto Deblur
software (version 6.001; AutoQuant Imaging, Watervliet, NY) was used.
Generation of IIGP1 and LRG-47 expression constructs
The coding regions of IIGP1 and LRG-47 were amplified either by PCR
from full-length cDNAs described in Ref. 6 from IFN-␥-stimulated mouse
embryonic fibroblasts according to standard procedures using PFU-polymerase (Promega, Madison, WI) and primers from Invitrogen Life Technologies. Restriction enzymes were from New England Biolabs (Beverly,
MA). The PCR fragments were cloned into the SalI site of pEGFP-C3
(Clontech, Palo Alto, CA), pEGFP-N3 (Clontech), pGW1H (British Biotechnology, Oxford, U.K.), or pGEX-4T-2 (Amersham Biosciences). Mutations of LRG-47 and IIGP1 were introduced into the open reading frames
according to the QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA) protocol. All constructs were verified by sequencing.
Test of induction of p47 GTPase in L929 and TIB-75 cells
L929 or TIB-75 cells were lysed at a concentration of 107 cell/ml in PBS,
1% Tx100 (Sigma-Aldrich) including complete mini protease inhibitors
(Roche, Basel, Switzerland), incubated for 1 h on ice, and centrifuged for
10 min at 23,000 ⫻ g at 4°C. Equivalent volumes were loaded onto 10%
polyacrylamide gels.
Membrane association assay of IIGP1-EGFP fusion proteins
Cosedimentation assay of rIIGP1
rIIGP1 was produced as previously described in Ref. 17. Liposomes were
prepared from synthetic phosphatidylserine (1,2-dioleoyl-sn-glycero-3-
Transfected L929 cells were lysed in hypotonic buffer (10 mM HEPES (pH
7.5), 5.1 mM MgCl2) 36 h after transfection as described above. After cell
disruption, lysates were centrifuged at 100,000 ⫻ g for 30 min at 4°C. The
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Golgi membranes (15); and LRG-47 has been copurified with phagosomes containing mycobacteria from infected macrophages (4). However, the intracellular localization of LRG-47 and the mechanism and
dynamics of membrane association of the p47 GTPases are unknown.
We show in this study that LRG-47 is normally localized to the
cis-Golgi by a C-terminal amphipathic helix. Upon phagocytosis,
LRG-47 is recruited locally to the plasma membrane, at the forming
phagocytic cups, and (in fibroblasts) also at phagocytosis-induced
membrane ruffles. This recruitment is likely to be mediated by sequences in the G domain and is probably nucleotide dependent.
LRG-47 then remains associated with the maturing phagosome,
reaching a late endosomal or lysosomal compartment colocalizing
with lysosome-associated membrane protein 1 (LAMP-1). A second
IFN-inducible member of the p47 GTPases, IIGP1, is localized to the
ER membrane, and this localization is mediated by mechanisms very
different from those guiding LRG-47 to the Golgi. However, like
LRG-47, IIGP1 also carries a cryptic targeting signal in its G domain
that can direct the free G domain to the plasma membrane. The related
but distinct cell biological behaviors of these two members of the p47
GTPase family are consistent with the idea that the family represents
a battery of cell-autonomous resistance factors diversified to engage
effectively with the variety of intracellular pathogens.
2595
2596
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
supernatant was stored on ice, and the pellet was washed as described
above and finally resuspended in PBS and 0.5% SDS. Equal amounts were
loaded on a 12% polyacrylamide gel.
Tx114 partitioning assay
L929 cells were lysed in PBS, 1% Tx114 (Sigma-Aldrich) including complete mini protease inhibitors (Roche) 24 h after IFN-␥ induction and 48 h
after transfection for 1 h on ice. Lysates were centrifuged for 15 min at
3,000 ⫻ g at 4°C to remove nuclei. Supernatants were incubated for 5 min
at room temperature and subsequently centrifuged for 1 min at 23,000 ⫻
g at room temperature. The aqueous phase was transferred into a new tube
and washed by an additional centrifugation step. The detergent phase was
washed by addition of PBS followed by an additional centrifugation step
and finally brought to the same volume as the aqueous phase by addition
of PBS. Equal amounts were loaded on a 12% polyacrylamide gel.
Transfections
Results
The IFN-inducible p47 GTPases show different levels of
membrane association
All five p47 GTPases tested were abundantly induced from very low
or undetectable resting levels in the mouse fibroblast-like cell line,
L929, and in TIB-75 mouse hepatocytes by IFN-␥. Expression of p47
GTPases was determined by Western blotting of Triton X-100 post
nuclear supernatants (Fig. 1a). IFN-␥-induced L929 cells were disrupted in a hypotonic buffer without detergent and centrifuged at
100,000 ⫻ g. Each p47 GTPase partitioned in characteristic ratio between pellet and supernatant fractions (Fig. 1b). LRG-47 was found
exclusively in the pellet fraction, whereas IRG-47 was largely in the
supernatant. IIGP1 partitioned roughly equally between the two
fractions.
LRG-47 localizes to the Golgi apparatus
L929 fibroblasts, TIB-75 hepatocytes, and RAW 264.7 macrophages were induced with IFN-␥ and stained for LRG-47. All the
cells showed a focused adnuclear signal accurately colocalizing
with Golgi markers Rab6 (20) (Fig. 2, a– c) and giantin (18) (d– g),
as well as showing a weaker diffuse signal throughout the cytoplasm (a, d, and h) partially colocalizing with the ER markers
calnexin and ERP60 (data not shown). No LRG-47 was detected in
cells not induced with IFN-␥ (Fig. 1, c and d). Less precise colocalization was seen with ␣-mannosidase II in RAW 264.7 cells (Fig. 2,
h–j) and no colocalization with the cation-independent mannose 6
phosphate receptor in L929 and RAW 264.7 cells (not shown) (21).
Therefore, LRG-47 seems to be associated with cis- and medial-Golgi
rather than with the trans-Golgi and the trans-Golgi network. We
could detect no colocalization of LRG-47 with the endocytic compartments defined by rab11 and endocytosed transferrin (not shown).
The localization of LRG-47 is different from that of its GMS relative
IGTP, which shows an exclusive ER association (14).
LRG-47 is recruited to the plasma membrane upon phagocytosis
To engage with pathogens entering via the phagosomal system (2,
4), LRG-47 might translocate to plasma membrane during phagocytosis. Peritoneal macrophages and the macrophage cell line
RAW 264.7 were induced with IFN-␥ for 24 h (Fig. 3). Phagocytosis of 2-␮m diameter latex beads was then followed by fluorescence microscopy for LRG-47 and F-actin. We observed a marked
FIGURE 1. Induction of p47 GTPases in L929 and TIB-75 cells and quantification of the soluble and membrane-bound fraction. a, Cells were stimulated with 200 U/ml IFN-␥ for 24 h and lysed in PBS containing 1% Triton
X-100. The equivalent of 105 cells was loaded in each track and subjected to
SDS-PAGE. b, L929 cells stimulated with 200 U/ml IFN-␥ for 24 h were lysed
in a hypotonic buffer followed by a 100,000 ⫻ g centrifugation step. Equal
amounts of the supernatant and pellet fractions were subjected to SDS-PAGE.
Proteins were detected by Western blot analysis using specific Abs or antisera.
c and d, L929 cells were induced with 200 U/ml IFN-␥ or left untreated for
24 h, fixed with 3% paraformaldehyde, and stained with the anti-LRG-47 A19
antiserum. Bound A19 was detected with a donkey anti-goat-Alexa 546 antiserum. In IFN-␥-induced cells, an adnuclear signal and a diffuse granular background throughout the cell fainting at the periphery was detected. No signal
was detected in noninduced cells.
recruitment of LRG-47 around forming phagocytic cups identified
by F-actin accumulation in proximity to latex beads in peritoneal
macrophages (Fig. 3, a– e) and in RAW264.7 macrophages (not
shown). Moreover, LRG-47 apparently remained associated with
the phagosomes as they matured to phagolysosomes, as demonstrated by the simultaneous labeling for LAMP-1 and LRG-47
(Fig. 3, f–j). To determine whether the phagocytosis-stimulated
relocalization of LRG-47 was a distinctive property of professional
phagocytes, we initiated phagocytosis in IFN-induced L929 fibroblasts using latex beads precoated with collagen, exploiting the
ability of fibroblasts to phagocytose collagen (22). Again, LRG-47
was found at F-actin-rich phagocytic cups and subsequently colocalized with the ingested beads in a LAMP-1-rich compartment
(Fig. 4, a–f). This colocalization was restricted to phagosomes
(Fig. 4, d–f); no colocalization of LRG-47 with LAMP-1 was seen
in cells not engaged in phagocytosis in L929 cells (g–i). The results of these experiments were essentially identical in L929 cells
and the two macrophage populations. Thus, the phagocytic process
itself appears more important as a stimulus for the relocalization of
LRG-47 than does identity of the membrane receptor or cell type
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Cells growing on heat-sterilized coverslips or cell culture dishes were transiently transfected with Fugene 6 (Boehringer, Ingelheim, Germany) according to the manufacturer’s protocol.
Prediction of secondary structure was done with programs found at
http://us.expasy.org/. The helical wheels were drawn with a program found
at http://marqusee9.berkeley.edu/kael/helical.htm.
The Journal of Immunology
2597
involved. Apparently, LRG-47 is recruited very early during the
phagocytic process, because LRG-47 could be detected at sites
where beads were attached to the cell surface before formation of
the phagocytic cup (Fig. 3, a– e).
IIGP1 is an ER-associated protein in fibroblasts, hepatocytes,
and macrophages
IIGP1 has been reported to colocalize with Golgi and to a lesser
extent with ER in mouse bone marrow-derived macrophages
(15), but the mechanism and dynamics of its membrane association are unknown. In contrast to LRG-47, IIGP1 partitions
between the cytosol and membrane compartments (Fig. 1b). We
analyzed the subcellular localization of membrane-bound IIGP1
in IFN-␥-treated L929, TIB-75, and RAW 264.7 cells, and fresh
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FIGURE 2. LRG-47 localizes to the
Golgi apparatus. L929 cells (a–c), TIB-75
cells (d–g), or RAW 264.7 (h–j) cells were
stimulated with 200 U/ml IFN-␥ for 24 h,
fixed, and stained for the indicated proteins
by indirect immunofluorescence. The distribution of LRG-47 largely overlaps with
rab6 in L929 cells (a–c). We also observed
overlap of LRG-47 with giantin and
Gm130 in L929 cells (not shown). In
TIB-75 cells, LRG-47 colocalized with giantin (d–g), although giantin staining is
spatially more restricted. Partial overlap of
LRG-47 and ␣-mannosidase II was observed in RAW 264.7 macrophages (h–j).
Nuclei were labeled with 4⬘,6⬘-diamidino2-phenylindole.
peritoneal macrophages. IIGP1 was detected in a reticular pattern throughout IFN-␥-induced cells, growing fainter at the periphery (Fig. 5, a, d, g, and j). No signal was seen in resting
cells. This pattern does not correspond with the proposed Golgi
localization previously reported for IIGP1 (15), but rather overlaps with ER proteins as shown for TAP (23) and calnexin in
peritoneal macrophages and ERP60 in TIB-75 cells (Fig. 5,
a–i). The most accurate colocalization with IIGP1 was observed
for TAP. We observed only insignificant colocalization with the
Golgi protein, giantin, in L929 cells and primary cultures of
peritoneal macrophages (not shown). Marginal, possibly significant colocalization with giantin was seen in RAW 264.7 macrophages (Fig. 5, j–m), although in this cell line, too, the vast
majority of IIGP1 localized in an ER-like reticular pattern.
2598
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
Therefore, we conclude that, at least in the cell types studied
here, IIGP1 is predominantly associated with the ER.
Golgi and ER association of LRG-47 and IIGP1 are
independent of GTP binding: plasma membrane binding of
LRG-47 requires bound nucleotide
To examine the nucleotide dependence of membrane binding by
LRG-47, a mutation analogous to that previously introduced
into IGTP (14) was generated, changing the sequence in the G1
motif from GX4GMS to GX4GMN. Both N-and C-terminal tags
cause mislocalization of LRG-47 to transferrin-positive recycling endosomes and other membranes of unknown identity (S.
Martens and J. C. Howard, unpublished results). Therefore, we
had to express untagged LRG-47 in a noninduced background.
For this purpose, wild-type and mutated forms of LRG-47 were
expressed in resting L929 cells and detected with A19 by indirect immunofluorescence. Wild-type LRG-47 localized to an
adnuclear region in a pattern indistinguishable from the induced, endogenously expressed LRG-47 (compare Figs. 6, a
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FIGURE 3. LRG-47 is recruited to
phagocytic cups and remains associated with the maturing phagosome in
macrophages. CB20-derived peritoneal
macrophages (a– e) or RAW 264.7
cells were induced with 200 U/ml
IFN-␥ for 48 h, incubated with 2-␮m
latex beads for 1 h, fixed, and stained.
LRG-47 is recruited very early during
phagocytosis. Close proximity of F-actin and LRG-47 could already been observed on not completely developed
phagocytic cups (c– e, middle arrow).
Colocalization of LRG-47 and
LAMP-1 was observed in peritoneal
macrophages (not shown) and RAW
264.7 cells (f–j). In RAW 264.7 cells,
the reticular distribution throughout the
cytoplasm is more pronounced than in
fibroblasts, hepatocytes, and peritoneal
macrophages. Nuclei were labeled with
4⬘,6⬘-diamidino-2-phenylindole.
and b, and 2) colocalizing with the Golgi proteins giantin and
Gm130 (24) (not shown). Apparently, positioning of LRG-47 in
the cell is independent of other IFN-␥-induced factors at physiological expression levels. When overexpressed at levels
higher than those induced by IFN-␥, however, LRG-47 translocated completely from endogenous membranes to the plasma
membrane leaving the Golgi region dark (Fig. 6, c and d). The
Golgi apparatus remains intact in these cells (Fig. 6, e and f).
When LRG-47 S90N was expressed in L929 cells, a staining
pattern similar to the wild-type protein expressed at normal
levels was observed (compare Fig. 6, g and h, with a and b),
although the reticular stain throughout the cell was noticeably
increased. The adnuclear signal colocalized with Gm130 and
giantin (not shown). However, unlike the wild-type case, no
localization to plasma membrane was seen even at the highest
expression levels of the mutant sequence. Therefore, we conclude that Golgi and ER membrane association of LRG-47 is
essentially independent of GTP binding, but plasma membrane
association is nucleotide dependent.
The Journal of Immunology
2599
When transfected into resting L929 cells, IIGP1 expressed at
low levels was localized in a reticular, ER-like pattern resembling the endogenous protein (not shown). However, cells
expressing higher levels tended to contain IIGP1 aggregates,
often of filamentous appearance, preferentially accumulating in
the adnuclear region (Fig. 6, i and j). Aggregates have not been
observed in any IFN-␥-induced cell line tested. Because IIGP1
forms GTP-dependent multimers in vitro (17), we asked
whether the formation of the observed accumulations requires
GTP-binding activity. A nucleotide-binding-defective IIGP1,
with the G1 motif mutated from GX4GKS to VX4VKS (17),
was expressed in L929 cells. Transfected IIGP1 G76, 81V was
associated with intracellular membranes but showed no aggregate formation regardless of expression level (Fig. 6, k and l).
The C terminus of IIGP1 has been reported to contribute to the
regulation of oligomerization of IIGP1 in vitro (17). Using the
recently determined crystal structure of IIGP1 (33), a deletion
mutant of IIGP1 was generated, lacking the whole C-terminal
domain following the F ␣ helix (␣F) (see Fig. 9a). In L929
cells, the truncated mutant IIGP1 aa 1–287 still bound to
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FIGURE 4. LRG-47 associates with
phagosomes. L929 cells were stimulated with 200 U/ml IFN-␥ for 20 h,
incubated with 2-␮m collagen-coated
latex beads coated with rat tail collagen
for 4 h, fixed, and stained for LRG-47
and for F-actin with Alexa 546-labeled
phalloidin (a– c). We detected association of LRG-47 with phagocytic cups
as determined by overlap with actin at
these sites. LRG-47 remained associated with the phagosomes as they matured to phagolysosomes visualized by
LAMP-1 stain (d–f). Colocalization of
LRG-47 with LAMP-1 was restricted
to phagosomes containing the collagen
beads (arrows). No colocalization was
seen in cells not engaged in phagocytosis (g–i). Nuclei were labeled with
4⬘,6⬘-diamidino-2-phenylindole.
intracellular membranes (Fig. 6, m and n), although the pattern
of localization was distinct from endogenous protein with a
marked accumulation on adnuclear endomembranes. Like the
G76, 81V mutant, no aggregates were observed regardless of
expression level. Thus, the C-terminal domain of IIGP1 is
essential for the formation of the aggregated structures seen in
transfected cells and contributes in some way to the specificity
of membrane association but is not required for membrane
binding as such.
IIGP1 is associated with the ER via N-terminal myristoylation
It is not known how the p47 GTPases bind to membranes. Of the
published p47 GTPases, only IIGP1 carries a predictable lipid
modification motif, namely an N-terminal myristoylation site
(within the N-terminal sequence MGQLFS, glycine 2 is predicted
to be myristoylated (25)). IFN-induced L929 cells were lysed in
Triton X-114, and the p47 GTPases IIGP1 and LRG-47 were assayed in the aqueous and detergent phases after temperature shift
(Fig. 7a). Consistent with lipid modification, ⬃50% of the IIGP1
2600
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
protein was found in the detergent phase despite the lack of wellmarked hydrophobic regions (6). In agreement with the lack of any
predicted lipid modification site, LRG-47 was found exclusively in
the aqueous phase (Fig. 7a), as were IGTP, TGTP, and IRG-47
(not shown). To determine whether the predicted N-terminal myristoylation site of IIGP1 is indeed used, we mutated glycine 2 to
alanine (G2A) and expressed the mutated protein in L929 cells
with wild-type IIGP1 as a control. IIGP1 G2A was found exclusively in the Triton X-114 aqueous phase (Fig. 7a). Transfected
wild-type IIGP1 partitioned in Triton X-114 like the endogenous,
IFN-␥-induced, protein.
To determine whether the N-terminal domain of IIGP1 is sufficient to target a soluble protein to membranes, we fused the Nterminal 68 aa, representing the N-terminal helical domain of
IIGP1 including ␣C, to the N terminus of enhanced GFP (EGFP),
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FIGURE 5. IIGP1 is associated with the ER in L929
cells, TIB-75 cells, and peritoneal macrophages, and
shows only minor Golgi association in RAW 264.7
macrophages. L929 cells (a– c), peritoneal macrophages (d–f), and TIB-75 (g–i) or RAW 264.7 cells
were stimulated with 200 U/ml IFN-␥ for 24 h, fixed,
and stained for the indicated proteins by indirect immunofluorescence. IIGP1 accurately colocalizes with
TAP (a– c). Colocalization was also observed with Calnexin in peritoneal macrophages (d–f) and ERP60 in
TIB-75 cells (g–i), although IIGP1 regularly showed a
more extended expression pattern than classical ER
proteins, indicating that IIGP1 might not be restricted
to subcompartments of the ER. j–m, Shown is an IFN␥-induced RAW 264.7 macrophage. The cell was
stained with anti-IIGP1 165 antiserum and anti-giantin
mAb. Sites of possible colocalization of IIGP1 and giantin are marked with arrows. Note that the majority of
IIGP1 does not colocalize with giantin. d–f and j–l,
Planes of 3D deconvoluted z-series are shown. Nuclei
were labeled with 4⬘,6⬘-diamidino-2-phenylindole.
expressed the construct in L929 cells, and lysed the cells in a
hypotonic buffer followed by ultracentrifugation. Partitioning into
the pellet and supernatant was monitored by Western blotting (Fig.
7b). EGFP alone was found exclusively in the supernatant. The
IIGP1 aa 1– 68 EGFP construct, in contrast, partitioned between
the supernatant and the pellet. The G2A mutant N terminus of
IIGP1 fused to EGFP was exclusively found in the supernatant,
suggesting that the N terminus of IIGP1 does not carry any myristoylation-independent membrane localization signal. These results were confirmed by immunofluorescence (not shown). The N
terminus is therefore sufficient for membrane association in a myristoylation-dependent manner. However, the myristoylated N terminus is not absolutely necessary for membrane association of
IIGP1 itself, in contrast to the N-terminal EGFP construct, because
the transfected full-length IIGP1 G2A mutant bound to membrane
The Journal of Immunology
2601
(Fig. 7, c and d), with only a small shift to the cytosolic compartment relative to wild-type IIGP1 (compare Figs. 1b and 7d). These
results suggest the presence of further membrane-targeting mechanisms in IIGP1.
Nonmyristoylated IIGP1 is a peripheral membrane protein
To determine the biochemical nature of the membrane association
of LRG-47 and IIGP1, we conducted a series of wash-off experiments (Fig. 8a). Isolated membranes were incubated in a variety of
different buffers, and partitioning of the p47 GTPases into the supernatant and pellet was determined by immunoblotting. LRG-47
and IGTP (not shown) behaved essentially like integral membrane
proteins such as Calnexin even in 100 mM sodium carbonate (pH
11.75). In contrast, IIGP1 and TGTP (not shown) were weakly
associated with membranes. The strength of membrane association
thus correlates with the classification into the GMS and GKS subgroups. The small amount of IIGP1 and TGTP in the Triton
X-100-insoluble fraction might represent aggregated protein or
possibly lipid raft- or caveolae-associated protein. Consistent with
the latter possibility, we observed some colocalization of IIGP1
with Caveolin-1 in TIB-75 hepatocytes (not shown). The wash-off
profile of IIGP1, and in particular the pronounced shift into the
supernatant for the sodium carbonate extraction, suggest that hydrostatic and ionic interactions dominate the binding of IIGP1 to
membranes. Therefore, we subjected membranes of L929 cells
transfected with the nonmyristoylated IIGP1 G2A mutant to the
same membrane extraction assay (Fig. 8a). As control, the washoff profile of wild-type IIGP1 expressed in resting L929 cells was
determined. Although the proportion of membrane-bound IIGP1
was significantly higher than in IFN-␥-induced cells, the wash-off
profile itself was not changed. Only a minor part of transfected
IIGP1 was Triton X-100 insoluble, indicating that the aggregates
observed in transfected L929 cells expressing high amounts of
IIGP1 (Fig. 6, i and j) are membrane-associated structures. Despite
the pronounced shift into the hypotonic supernatant noted above,
the residual membrane-bound IIGP1 G2A showed the same washoff profile as the transfected wild-type protein (Fig. 8a). This suggests that the myristoyl group regulates access to membrane rather
than conferring strong membrane binding. Nevertheless, the N terminus of IIGP1 is able to target EGFP to membranes in a myristoyl-dependent manner (Fig. 7b).
The binding of IIGP1 to membranes independent of lipid modification might be mediated by protein-protein interactions or by a
direct interaction with lipids. To distinguish between these
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FIGURE 6. Membrane association of LRG-47 and IIGP1 is independent of nucleotide binding. L929 cells were transfected with the indicated expression
constructs, fixed 24 h thereafter, and stained for LRG-47 or LRG-47 and giantin. The localization of transfected LRG-47 is independent of other
IFN-␥-induced factors (a and b). The transfected LRG-47 colocalized with giantin and Gm130 (not shown). When massively overexpressed, LRG-47 was
found to be exclusively associated with plasma membrane (c and d). The Golgi apparatus remains intact in L929 cells expressing high amounts of LRG-47
(e and f). LRG-47 S90N has a distribution very similar to that of the wild-type protein (g and h), although the non-Golgi cytoplasmic signal was relatively
increased, overlapping with Calnexin and ERP60 (not shown). IIGP1 forms nucleotide- and C-terminal domain-dependent aggregates in resting L929 cells
and associates with membrane independently of nucleotide binding. L929 cells were transfected with the indicated expression constructs, fixed 24 h later,
and stained with 165 anti-IIGP1 antiserum. Cells expressing IIGP1 showed IIGP1-containing enigmatic structures often apparently filamentous in character
(i and j). A nucleotide-binding-deficient mutant (G76, 81V) was still capable of membrane association, but no IIGP1-containing structures were seen
regardless of expression level (k and l). Deletion of the C-terminal domain abolished the ability to induce intracellular aggregates but not membrane
association (m and n). c, A plane of a 3D deconvoluted z-series is shown.
2602
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
alternatives, we analyzed the lipid-binding properties of purified,
bacterially expressed IIGP1 (17) in vitro. The purified protein has
a nonhydrophobic N-terminal extension of 10 aa derived from the
GST-fusion (17) and is not myristoylated. When incubated with
artificial unilamellar 100-nm phosphatidylserine vesicles, rIIGP1
efficiently cosedimented with the lipids (Fig. 8b). The wash-off
profile of this protein is very similar to that of the in vivo-expressed protein (Fig. 8c). This result strongly suggests that IIGP1
has an intrinsic ability to interact with lipids, and that this interaction is independent of other proteins. A variable quantity of Triton X-100-resistant, presumably denatured, aggregated, protein
FIGURE 8. The p47 GTPases
show different membrane association
properties. a, L929 cells were stimulated with 200 U/ml IFN-␥ or transfected with the indicated constructs
and lysed in a hypotonic buffer 48 h
later. Membranes were prepared and
resuspended in the indicated buffers.
After centrifugation, membranes were
dissolved in PBS, 0.5% SDS. Equal
volumes were subjected to Western
blotting. b, Purified, nonmyristoylated
rIIGP1 was incubated with synthetic
phosphatidylserine vesicles at room
temperature and spun at 100,000 ⫻ g.
Equal volumes of the supernatant and
pellet fractions were subjected to
Western blotting. c, rIIGP1 was bound
to lipids and processed as in a. The
wash-off profile and thus the membrane-binding properties of rIIGP1 are
very similar to the endogenous and
transfected wild-type and G2A mutant
IIGP1.
was seen with rIIGP1 incubated with artificial phosphatidylserine
vesicles (Fig. 8c).
Individual domains contribute independently to the targeting of
the p47 GTPases to membranes
To guide a systematic analysis of the membrane-targeting signals
of IIGP1 and LRG-47, we exploited the recently determined structure of IIGP1 (33). The linear organization of the p47 GTPases is
shown in Fig. 9a. Helices in the N- and C-terminal parts are shown
as lines. Secondary structure elements in the G domain are not
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FIGURE 7. IIGP1 but not LRG-47 is lipid modified, showing myristoylation-dependent membrane-targeting activity. L929 cells were stimulated with
200 U/ml IFN-␥ or transfected with the indicated expression constructs, harvested after 24 h, and extracted with cold Triton X-114 (a), lysed in a hypotonic
buffer (b and d), or fixed (c). a, After phase separation at room temperature, equal volumes of the aqueous and detergent phase were subjected to
SDS-PAGE. After blotting, the membrane was cut, and the upper part was incubated with anti-IIGP1 165 antiserum and the lower part with N20
anti-Caveolin-1 antiserum, serving as load control. The same IFN-␥-induced cell lysates were loaded on a separate gel to detect LRG-47. LRG-47 was
exclusively found in the aqueous phase, whereas IFN-␥-induced as well as transfected IIGP1 partitioned between both phases. Mutating the N-terminal
myristoylation motif of IIGP1 by replacing glycine 2 with alanine resulted in complete shift into the aqueous phase. b, The first 68 aa of wild-type IIGP1
but not of the G2A mutant target EGFP to intracellular membranes. The full-length IIGP1 G2A mutant, in contrast to the mutant N terminus, still bound
to membrane (c and d).
The Journal of Immunology
An amphipathic helix near the C terminus is responsible for
Golgi targeting of LRG-47
Although the plasma membrane association of the G domains is
intriguing, it does not reflect the steady-state localization of the
p47 GTPases observed in IFN-␥-induced cells. A good candidate
for the distinctive Golgi membrane targeting of LRG-47 is the
C-terminal domain, which is a highly divergent region of the p47
GTPases (6). The C-terminal domains of IIGP1 and LRG-47 were
fused to the C terminus of EGFP and expressed in L929 cells. For
LRG-47, the C terminus was fully active in targeting EGFP to the
Golgi apparatus (Fig. 9, d–f). In addition, however, we observed a
marked, diffuse, reticular stain throughout the cell similar to
LRG-47 S90N (compare with Fig. 6g). However, the C terminus of
IIGP1 does not confer membrane association by itself, because the
EGFP fusion construct appeared fully soluble (Fig. 9k). Thus,
LRG-47 and IIGP1 use different domains to bind to membranes.
To identify the Golgi membrane-targeting motif in the C-terminal
domain of LRG-47, we made a series of N-terminal truncation
mutants of the C-terminal domain (Fig. 9b and not shown). These
were fused to the C terminus of EGFP and expressed in L929 cells.
All LRG-47 constructs containing the region corresponding to the
last two helices, ␣K-L, of IIGP1 showed membrane association
and Golgi localization (not shown). Further truncating ␣K and the
loop preceding ␣L led to complete loss of membrane association
(not shown). The deleted region between the last two mutations
represents 25 aa and includes an ␣ helix in IIGP1 that is also
predicted in LRG-47 2 (␣K (Fig. 9, a and b)) (33). These 25 aa of
LRG-47 (Fig. 9b) were fused to the C terminus of GFP and expressed in L929 cells. Apparently, this small fragment was fully
competent to target GFP to Golgi (Fig. 9, g–i). In LRG-47 (but not
in IIGP1), this putative helix (aa 356 –369) has an amphipathic
character when drawn as a helical wheel (Fig. 9c). Therefore, we
inserted glutamate residues at several positions that are predicted
to destroy the amphipathicity of helix K (Fig. 9c). When the ␣K
fragment with the insertions (ins 362, 367E) was expressed in
L929 cells, membrane binding and Golgi association were com-
pletely abolished (Fig. 9j). The same result was obtained for the
whole C-terminal domain for which all three mutations shown
(Fig. 9b) completely abolished membrane association (not shown).
To examine the effect of the ␣K mutations on the distribution of
the whole molecule, full-length LRG-47 carrying these mutations
was expressed in resting L929 cells. Unlike the isolated C-terminal
fragment containing these mutations, the full-length LRG-47 protein
was still membrane bound. However, in contrast to wild-type LRG-47
(Fig. 9n), none of the ␣K mutants accumulated on Golgi membranes
(o). Thus, LRG-47 is targeted to Golgi membrane by an amphipathic
helix located in the C-terminal region at a position corresponding to
␣K in the structure of IIGP1. In the crystal structure of IIGP1, the ␣K
helix is positioned on the same surface as the N terminus carrying the
myristoylation motif. If this location is conserved in LRG-47, the
orientation of the two GTPases toward the membrane would be similar despite the different membrane attachment mechanisms.
Discussion
The p47 GTPases are critically important molecules in the mouse
for resistance against intracellular pathogens, but very little is
known about how they work. One member of the family, LRG-47,
mediates resistance against L. monocytogenes (2), T. gondii (2), M.
tuberculosis (4), and M. avium (5). Phagocytosis is a common
mode of entry for pathogens of radically different biologies. However, acidification of the phagosomal vacuole and the activation of
lysosomal hydrolases usually makes the compartment uninhabitable soon after particle uptake. Therefore, many intracellular
pathogens that are taken up by phagocytosis try to delay or avoid
acidification and the targeting of the phagosome to lysosomes (26).
The finding that LRG-47-deficient macrophages showed a measurable defect in acidification of Mycobacterium-containing
phagosomes (4) suggested that LRG-47 might counteract such survival strategies by marking the phagosome for rapid maturation, as
proposed for the IFN-inducible Rab GTPase, Rab5a, which confers resistance to L. monocytogenes (27). However, in this study,
we show that LRG-47 in the IFN-induced cell is not normally a
protein of the endolysosomal compartment. Rather, it localizes
mainly to the cis-Golgi, with weak expression on the ER (Fig. 2).
Recruitment of LRG-47 to regions of actin polymerization at the
plasma membrane, including phagocytic cups, is apparently initiated by phagocytosis, and the protein then remains associated with
phagosomes as they mature to the lysosomal compartment (Figs. 3
and 4). The transfer of LRG-47 to sites of phagocytosis occurs
within minutes of addition of latex beads. There is no detectable
cytosolic pool of LRG-47 (Fig. 1). Therefore, the protein either
must be rapidly and transiently released from Golgi (or possibly
ER) membranes before reassociating with the plasma membrane,
or else the relocalization is achieved by induced fusion of Golgi
(or possibly ER) membrane with active regions of plasma
membrane (28).
The relocation of LRG-47 from Golgi to actin-rich plasma
membrane is not restricted to professional phagocytes; LRG-47
behaved in essentially the same way in fibroblasts and macrophages (Figs. 3 and 4). Moreover pathogen-specific signals are
clearly not required to induce LRG-47 relocalization, because in
our experiments phagocytosis was initiated with uncoated (macrophages and RAW246.7 cells) and collagen-coated (L929 cells)
latex beads. Instead, processes associated with actin remodeling,
rather than phagocytosis as such, are more plausible triggers. Consistent with this suggestion, LRG-47 was also localized at actinrich plasma membrane ruffles adjacent to the forming phagosomes
in L929 cells, as well as at phagocytic cups themselves (Figs. 3 and
4). However, it will be important to determine the recruitment of
LRG-47 and the other p47 GTPases to plasma membrane and
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shown (33). The numbers refer to the amino acid positions in the
primary structure of LRG-47 and IIGP1, respectively. As shown in
Fig. 7, the N-terminal domain does not account fully for the membrane-binding properties of IIGP1 and is not responsible for membrane association of LRG-47. To localize further membrane-targeting regions of the two GTPases, the isolated G domains, the G
domains with the C-terminal ␣E, ␣F helical extensions (Fig. 9a),
as well as the C-terminal domains of IIGP1 and LRG-47, were
expressed in L929 cells. The first two fragments were tagged with
the FLAG epitope tag at the C terminus, whereas the last was fused
in frame to the C terminus of EGFP. Transiently transfected L929
cells were fixed 24 – 40 h after transfection and stained with the
indicated Abs. Strikingly, the isolated G domain of IIGP1 showed
exclusive plasma membrane association (Fig. 9l). The isolated G
domain of LRG-47 was not expressed in L929 cells. Therefore, we
expressed a construct encoding the G domain of LRG-47 extended
at the C terminus to include the ␣F helix. This construct also showed
exclusive plasma membrane binding (Fig. 9m) indistinguishable from
the isolated G domain of IIGP1 (l). No internal membranes were
stained. This localization recalls the plasma membrane recruitment of
LRG-47 upon phagocytosis of latex beads (Figs. 3 and 4) and possibly
implicates the G domain in redirecting LRG-47 to the plasma membrane. The plasma membrane staining is particularly marked at
plasma membrane ruffles for both LRG-47 and IIGP1, again similar to
the observed recruitment of LRG-47 to membrane ruffles in L929
cells engaged in phagocytosis.
2603
2604
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
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FIGURE 9. (Legend continues)
The Journal of Immunology
2605
Table I. Summary of the membrane association properties of the mouse p47 GTPases
Localization
% Membrane
Bound
Mechanism of Membrane Association and
Targeting
Nucleotide Dependence of
Membrane Association
Reference
⬎90%
Amphipathic helix in the C terminus,
Unidentified signal in the G domain
Nucleotide independent
Nucleotide dependent
This study; 4
This study
IGTP
IIGP1
Golgi, ER,
Plasma membrane,
phagosome
ER
ER, Golgi
⬎90%
⬃60 –70%
Nucleotide independent
Nucleotide independent
15
This study; 16
TGTP
IRG-47
ND
ND
⬃20 –30%
⬍10%
ND
N-terminal myristoylation, unidentified
signal in the G domain
ND
ND
ND
ND
This study
This study
LRG-47
LRG-47
regulated exchange of Arf1 between cytosol and membranes,
which also entails the GTP-dependent repositioning of an amphipathic helix (31, 32). The role of nucleotide in the membrane positioning of the isolated LRG-47 G domain is presently under direct investigation.
The propensity to bind to plasma membrane may be a general
feature of the p47 GTPases and not just of LRG-47, because the
phylogenetically rather distinct IIGP1, a member of the GKS subfamily of p47 GTPases, also has plasma membrane-targeting activity in the conserved G domain (Fig. 9l). However, in other respects, the differences between IIGP1 and LRG-47 are more
conspicuous than the similarities. Thus, the data presented above
establish IIGP1 to be an ER-associated GTPase in all cell types
analyzed (Fig. 5), unlike LRG-47. In this respect, our results appear to differ from those of Zerrahn et al. (15), who reported predominantly Golgi localization of IIGP1 in bone marrow-derived
macrophages. Native IIGP1 is targeted to membranes by N-terminal myristoylation (Fig. 7). The myristoylation signal is present
only on IIGP1 among the published p47 GTPases, but of the 23
p47 GTPase genes present in the mouse genome, 9 are predicted to
encode myristoylated proteins (C. Bekpen, J. Hunn, L. Guethlein,
D. M. Dunn, E. Glowalla, I. Parvanova, and J. C. Howard, manuscript in preparation). Therefore, IIGP1 is probably representative
of a large subfamily of p47 GTPases in the mouse, at least with
respect to its membrane targeting. However, myristoylation is not
the only membrane attachment mechanism in IIGP1. Full-length
IIGP1 protein mutated at the myristoyl attachment glycine, G2,
was still ⬃40% membrane bound in transfected cells and distributed on endomembranes (Fig. 7). Because the isolated G domain
targets the plasma membrane, not endomembranes, there must be
a further site of interaction with endomembranes elsewhere in the
molecule. Unlike LRG-47, however, the whole C-terminal region
of IIGP1 lacked membrane-targeting activity when fused to EGFP.
It is presently unclear where the residual membrane interaction
FIGURE 9. The localization of LRG-47 and IIGP1 is controlled by different domains. a, The domain structure of IIGP1 and LRG-47 based on the crystal
structure of IIGP1 is shown (33). The numbers indicate the positions within the protein sequence of LRG-47 and IIGP1, respectively. ␣ Helices in the
N-terminal and C-terminal domains are shown as small lines. Isolated domains were fused to EGFP or FLAG tagged. b, An enlarged scheme of the C
terminus of LRG-47 and three shorter constructs designed to map the membrane-targeting sequence in LRG-47 are shown. L929 cells were transfected with
the indicated constructs, fixed, and stained with the indicated Abs after 24 h. d–f, Shown are two L929 cells transfected with the C-terminal domain of
LRG-47 fused to the C terminus of EGFP. The fusion protein accurately localized to the Golgi apparatus as shown by the costain with giantin. Some diffuse
peripheral stain was also apparent. The C-terminal domain of IIGP1 does not bind to membrane (k). To map the targeting sequence, smaller fusion
constructs were expressed in L929 cells (b). The smallest membrane-bound construct that still localized to the Golgi apparatus included ␣K and ␣L (not
shown) comprising 59 aa. Further deleting 25 aa, comprising ␣K and the loop preceding ␣L, led to complete loss of membrane binding (not shown). When
fused to GFP, these 25 aa accurately localized the fusion construct to the Golgi apparatus (g–i). Secondary structure predictions of the deleted 25 aa
consistently predicted an ␣ helix. A core region starting from aa 356 forms an amphipathic helix when drawn as a helical wheel (c). The amphipathic
character of the helix was destroyed by inserting a glutamate at position 359, 362, or 362 and 369 (c) into the 25-aa-long ␣K fragment fused to EGFP,
leading to complete loss of Golgi targeting (j). When introduced into the full-length open reading frame, the ␣K mutations led to loss of Golgi accumulation
(n and o) in contrast to the wild-type (wt) LRG-47 protein (arrows) but not of membrane binding. The isolated G domain of IIGP1 and the G domain plus
␣E and ␣F in the case of LRG-47 localize to plasma membrane in transiently transfected L929 cells (l and m). Cells were stained with anti-FLAG M2 Ab
to detect the C-terminal-tagged G domain fragments and with A19 to detect the full-length wt and insertion (ins) 362, 367 LRG-47 molecules.
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phagosomes containing pathogens, because this process is significantly more complex than the uptake of latex beads (29).
The mechanism of LRG-47 recruitment from Golgi or ER to
plasma membrane by phagocytosis is still unknown. However, the
recent report of selective activation of Ha-Ras on Golgi membrane
by signals emanating from plasma membrane provides an interesting precedent (30). There is unfortunately no information about
regulated nucleotide exchange in p47 GTPases and no clear definition of what activation of these GTPases really means (17). Nevertheless, our present data suggest that regulated nucleotide binding may be relevant to the positioning of LRG-47 in the cell.
Overexpression unexpectedly caused transfected wild-type
LRG-47 to relocate to the plasma membrane and particularly to
membrane ruffles, completely excluding Golgi membranes (Fig. 6,
c and d). Interestingly, however, this behavior was dependent on
the integrity of the nucleotide binding site (Fig. 6, g and h). Therefore, it is not implausible that phagocytosis-stimulated repositioning of LRG-47 caused by a signal initiated at the membrane may
also entail a nucleotide-dependent modification of the GTPase.
The Golgi localization of LRG-47 is mediated by a C-terminal
amphipathic helix (Fig. 9). When this is disrupted, transfected
LRG-47 is localized on internal membranes but not on the plasma
membrane (Fig. 9, n and o). Thus, plasma membrane localization
is not a simple default state. However, the isolated G domain of
LRG-47 stripped of the N- and C-terminal helical domains localizes constitutively to the plasma membrane. In the IIGP1 structure,
the wild-type C terminus region interacts with the G domain (33)
and is required for accelerated GTP hydrolysis (17). Loss of the
C-terminal domain of LRG-47 may thus delay GTP hydrolysis in
vivo, causing the protein to behave like a constitutively active
GTPase. If LRG-47 is released from internal membranes before
transfer to actin-rich plasma membrane, then the interaction of at
least one and possibly two amphipathic helices with the membrane
must be disrupted. This behavior would then be reminiscent of the
2606
DYNAMIC ASSOCIATION OF p47 RESISTANCE GTPases WITH MEMBRANES
Acknowledgments
We acknowledge our debt to Dr. Ulrich Boehm for his contributions to the
initiation of this work and are grateful to Prof. Klaus Pfeffer for his continuing
support and interest in the project. We are also grateful to the many colleagues
who provided serological reagents (referred to individually in Materials and
Methods) and to Dr. Matthias Cramer, who performed the immunizations and
bleedings that resulted in anti-IIGP1 serum 165. We acknowledge the collaboration with Dr. Agnidipta Ghosh and Prof. Christian Herrmann of the Max
Planck Institute for Molecular Physiology, which resulted in the solution of the
crystal structure of IIGP1. We are indebted to Dr. Jens Zerrahn (Max Planck
Institute for Infection Biology, Berlin, Germany) for his generous gift of the
10D7 and 10EY mAbs directed against IIGP1. Prof. Maria Leptin made helpful comments on the manuscript.
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sites of IIGP1 are. Also, unlike LRG-47, IIGP1 has a substantial
cytosolic pool in the IFN-induced cell. The results point to IIGP1
behaving as a classical peripheral membrane protein with relatively weak interactions with the membrane. This conclusion was
fully confirmed by the relative ease with which IIGP1 could be
released from the membrane fraction of disrupted cells. In contrast,
LRG-47 behaved in this assay in all respects like a transmembrane
protein, despite partitioning in the aqueous phase in Triton-X114
(Figs. 7 and 8).
The residual membrane-binding behavior of demyristoylated
IIGP1 in vivo could be reproduced by purified bacterially extracted
IIGP1 binding to synthetic phosphatidylserine lipid vesicles in
vitro (Fig. 8, b and c). This result suggests that IIGP1 may also
interact directly with membrane lipids in the cell, to some degree
independently of the local protein environment. In this respect,
IIGP1 is similar to LRG-47, where the strong interactions with
membrane appear to be based not on protein-protein interactions,
but on the C-terminal amphipathic helix and possibly a second
similar interaction with lipid that we have not yet located. The
ability of p47 GTPases to bind to membranes exclusively via lipids
may have some adaptive importance, because intracellular pathogens such as Toxoplasma commonly modify the protein compositions of their compartments to avoid destructive host processes
such as phagosome maturation.
The diversity of intracellular behavior of the p47 GTPases is
very striking. Among the small number studied here, the GKS
group (IIGP1, TGTP, and IRG-47) have markedly looser association with intracellular membranes than the GMS group (IGTP and
LRG-47). Furthermore, they are associated with different intracellular compartments even within a group. Thus, LRG-47 is basically a protein of the cis-Golgi membrane, whereas IGTP is an ER
membrane protein. Neither has a significant cytosolic pool.
IRG-47 seems to be primarily a cytosolic protein, whereas TGTP
and IIGP1 also have large cytosolic pools (Table I). As we have
seen, LRG-47 and IIGP1 use radically different means of associating with membranes. It is already clear that different members of
the family have different profiles of activity against specific pathogens (3). The biochemical differences shown in this study, which
are reflected in strikingly divergent amino acid sequence, will
surely prove to be critical parameters determining the nature and
mechanism of the resistance functions exercised by the p47
GTPases. It is remarkable that the existence of these powerful
mediators of cell-autonomous immunity was unknown for so long.