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Microbiology
Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts
Departments of *Biochemistry and Microbiology,
University of Connecticut Health Center,
263 Farmington Avenue,
Farmington, CT 06032 USA
Communicated by M. J. Osborn, University of Connecticut Health Center, Farmington, CT and approved September 30, 2002 (received for review August 23, 2002)
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Abbreviations: ARF1, ADP-ribosylation factor-1; BsMinD, Bacillus subtilis MinD; EcMinD, Escherichia coli MinD; MTS, membrane-targeting sequence
MinD is the best conserved and most widely distributed of the Min proteins, being found in all domains of life (eubacteria, archaea, and eukaryotes). It is a member of the ParA superfamily of ATPases, most of which (apart from the MinD subgroup) are involved in plasmid or chromosome partitioning (10–12). The ATPase activity of MinD is presumed to provide the driving force for pole-to-pole oscillation of the MinC-MinD division inhibitor (13, 14). This activity is stimulated by MinE but only in the presence of phospholipids (13, 14). MinD is a peripheral membrane protein (10), and its association with the inner membrane is a prerequisite for subsequent recruitment of both MinC and MinE to the membrane. However, the mechanism by which MinD associates with the inner membrane and subsequently recruits MinC and MinE remains enigmatic; it seems to lack a transmembrane domain, and the amino acid sequence is devoid of hydrophobic stretches that might predispose the protein to interaction with lipids. It has been suggested that MinD might be localized to the inner membrane by means of interactions with a membrane protein (15). However, recent studies have demonstrated conclusively that, at least in vitro, MinD is capable of binding directly to lipid bilayers (13, 14). Furthermore, this interaction induces extensive MinD polymerization (14).
Crystal structures have been determined for presumptive MinD homologs from the hyperthermophilic archaeons Archaeoglobus fulgidus (15), Pyrococcus furiosus (16), and Pyrococcus horikoshii (17). Unfortunately, these structures provide little insight into the mechanism by which MinD associates with the membrane. Intriguingly, however, all three structures are incomplete in the C-terminal region. No electron density was observed for the C-terminal 30 and 8 residues of A. fulgidus and P. furiosus MinD, respectively, presumably because of the high intrinsic flexibility of these residues. Although electron density was obtained for all but the final two residues in the crystal of P. horikoshii MinD, the C-terminal residues 235–243 had very high B factors (17), indicative of significant motion in the crystal. On the basis of these crystal structures, we speculated that the C-terminal region of MinD might be involved in membrane attachment in vivo, and that this region might be structured only when associated with lipid. In this study, we show that the C-terminal region of MinD, but not that of other members of the ParA superfamily, contains a highly conserved motif that is essential for membrane localization. We propose that this motif forms an amphipathic helix that mediates a direct interaction between MinD and membrane phospholipids.
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Experimental Procedures |
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19,
EcMinD5, EcMinD3, and
EcMinD2) were generated by PCR using pSLR22 as the
template and primers that incorporated flanking
XbaI/HindIII restriction sites. An
EcMinD(L267E) point mutant and several insertion mutants
were generated in a similar manner by using 3' primers incorporating
the desired mutations.
Bacillus subtilis MinD (BsMinD) was
obtained by PCR using the full-length minD gene as template
and 5' and 3' primers incorporating XbaI and PstI
restriction sites, respectively. This construct was then ligated, along
with a PCR-generated GFPmut2 gene incorporating 5'
EcoRI and 3' XbaI sites, into plasmid pSJ4 (18)
that had been digested with EcoRI/PstI. This
ligation yielded plasmid pTS26 in which BsMinD is
encoded as in-frame fusion to the C terminus of GFP. Various C-terminal
truncation mutants (BsMinD24, BsMinD5,
BsMinD4, BsMinD3, and
BsMinD2) were then generated by PCR using pTS26 as the
template and primers that incorporated flanking
XbaI/PstI restriction sites.
Fluorescence Microscopy.
For each GFP-fusion construct, cells from a single colony were
grown in LB supplemented with 20 µg/ml chloramphenicol and
1% glucose at 37°C for 4 h (OD600
0.5–0.7). Harvested cells (1 ml) were washed in medium lacking
glucose before inoculating fresh LB medium containing 20 µg/ml
chloramphenicol and 0.005% arabinose to a starting
OD600 of 0.05. Cultures were grown at 30°C for
4–5 h. Cells were then viewed live and after fixation for 45 min in a
final concentration of 1.7% (vol/vol) formaldehyde and 0.17%
(vol/vol) glutaraldehyde. Cells were viewed by using an Olympus BX40
microscope (Olympus, New Hyde Park, NY), and images were captured by
using a MagnaFire digital camera (Optronics International,
Chelmsford, MA).
Western Blot Analysis. Cells collected from fluorescence experiments were pelleted, resuspended in 0.5% SDS, boiled for 5 min, then stored at –80°C until use. The total protein content of each strain was determined by bicinchoninic acid assay (Pierce). SDS/polyacrylamide gels were loaded with 5 µg of total cell protein for each GFP-fusion construct. The gel was electroblotted onto Hybond ECL nitrocellulose membrane (Amersham Pharmacia), and GFP-fusion proteins were probed by using rabbit anti-GFP primary antibodies (Molecular Probes) and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia). Immunoblots were developed on Hyperfilm ECL (Amersham Pharmacia) by using a chemiluminescent substrate.
Overexpression and Purification of MinD.
An NdeI–BamHI PCR fragment encompassing
the entire E. coli minD gene was cloned into
pET-15b (Novagen) to produce plasmid pTS21, in which EcMinD
is encoded as an N-terminal His6-tag fusion
protein. His6-EcMinD was overproduced
by expression from pTS21 in E. coli
BL21minCDE:kan(
DE3) cells (a kind gift
from D. RayChaudhuri, Tufts University, Boston). An
NdeI–EcoRI PCR fragment encompassing the first
251 codons of the E. coli minD gene followed by a
stop codon was cloned, in-frame, into pET28a to produce the plasmid
pSLR90, in which EcMinD19 is encoded as an N-terminal
His6-tag fusion protein. Expression of
His6-EcMinD19 and
His6-EcMinD in either BL21(DE3) or
its minCDE derivative, respectively, was induced with 1
mM isopropyl 
-D-thiogalactoside after
growth at 34°C to an OD600 of 0.6. Cells were
grown at 30–34°C for a further 3 h before being harvested by
centrifugation, resuspended in binding buffer (5 mM imidazole/500 mM
NaCl/20 mM Tris•HCl, pH 7.9), and lysed by using a French press.
MinD fusion protein was purified from the soluble fraction by nickel
affinity chromatography with His-bind resin (Novagen). Washes with
60–120 mM imidazole were used to elute contaminating proteins.
His6-EcMinD (or
His6-EcMinD19) was then eluted by
using elution buffer (190 mM imidazole/500 mM NaCl/20 mM
Tris•HCl, pH 7.9) and dialyzed against appropriate buffer in
preparation for CD spectroscopy. Final protein preparations were judged
from Coomassie blue-stained SDS/polyacrylamide gels to be
>98% pure.
CD Spectropolarimetry. CD spectra were recorded at 4°C by using a Jasco J-715 spectropolarimeter (Jasco, Easton, MD). Protein samples were 10 µM in 1 mM NaPi/20 mM NaF, pH 7.9. Spectra were the average of 16 scans acquired by using a scan rate of 20 nm•min–1 and a response time of 8 s.
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-helix (designated 11 in the two Pyrococcus
structures) (16, 17). Sequence alignments show that the C terminus of
11 corresponds to Leu-247 of E. coli MinD. Thus, we
initially examined the localization of a mutant EcMinD
truncated by 19 residues at Arg-251; this conservative truncation
allows for the possibility that 11 might be slightly longer in the
E. coli protein.
Removal of the C-terminal 19 residues yielded a mutant
(GFP-EcMinD19) that failed to exhibit a peripheral
localization pattern (Fig. 1D). Instead, the cells appeared
uniformly green, suggesting that at least some portion of the
C-terminal 19 residues is essential for membrane localization of
EcMinD. To delineate better the portion of the C terminus
contributing to membrane localization, shorter MinD truncation mutants
were generated. A GFP-EcMinD5 truncation mutant failed to
localize properly (Fig. 1E), indicating that key
determinants for membrane localization exist in the C-terminal five
residues of EcMinD. Significantly, a two-residue C-terminal
truncation (GFP-EcMinD2) localized normally to the
periphery of the cell (Fig. 1G), whereas a three-residue
C-terminal truncation (GFP-EcMinD3) was diffusely
distributed throughout the cytoplasm (Fig. 1F). This
finding indicates that the third residue from the C terminus (Phe-268)
is critical for attachment of EcMinD to the inner membrane,
whereas the two C-terminal glycine residues are dispensable for this
function.
Western blot analysis of cells expressing each of the
GFP-EcMinD truncation mutants indicated that the MinD
protein levels were similar to those observed in cells expressing WT
GFP-EcMinD (Fig. 1H). Furthermore, the CD
spectrum of purified His6-EcMinD19
was very similar to that of
His6-EcMinD (Fig.
1I), indicating that deletion of the C-terminal 19
residues does not perturb the global fold of the protein. The high
thermal stability of the protein was also unaffected by deletion of the
C-terminal 19 residues; the thermal unfolding temperature
(Tm) of both EcMinD19
and EcMinD was >90°C (CD data not shown). Because 19
was the largest deletion studied, it is safe to assume that the smaller
truncations (2, 3, and 5) also have no effect on the global
fold or thermodynamic stability of MinD. We conclude that the aberrant
localization observed for the 3, 5, and 19 truncation mutants
was not due to an effect on the stability or global fold of the protein
but rather was due to the loss of key determinants for membrane
localization that are present in the C-terminal region of
EcMinD.
A Conserved Membrane-Targeting Sequence in the C-Terminal Region of MinD. Although MinD is the best conserved of the Min proteins, the highest levels of sequence conservation are restricted to the Walker-type ATPase motifs located in the N-terminal region of the protein. However, close inspection of the C-terminal region reveals a short sequence motif that is phylogenetically well conserved (Fig. 2). Although the 10-residue sequence spanning residues Lys-261–Gly-270 of the E. coli sequence is remarkably well conserved among Gram-negative eubacteria, we showed that the terminal diglycine sequence is dispensable for membrane localization of EcMinD, and the sequence alignment in Fig. 2 shows that these residues are not highly conserved outside of Gram-negative bacteria. Thus, we propose that a slightly more restricted sequence motif corresponding to Lys-261–Phe-268 of EcMinD is responsible for membrane localization of MinD in eubacteria, archaea, and plastids. We refer to this as the MinD membrane-targeting sequence (MTS).
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min strain PB114. As shown in Fig.
3A, GFP-BsMinD
exhibited a distinct peripheral localization pattern extending around
the entire circumference of the cell; the pattern of localization in
E. coli was indistinguishable from that of
GFP-EcMinD (Fig. 1C). To confirm that this
localization was indeed due to the proposed MTS, we examined the
localization of several truncation mutants (Fig. 3E). First,
we examined the localization of GFP-BsMinD24 in which the
C-terminal 24 residues of BsMinD were removed.
BsMinD contains a small insertion in the C-terminal region
compared with EcMinD, as well as a longer extension beyond
the proposed MTS (see Fig. 2), so BsMinD24 effectively
corresponds to the EcMinD19 truncation mutant.
GFP-BsMinD24 failed to exhibit a peripheral localization
pattern (Fig. 3B), indicating that key determinants for
membrane localization are located, as predicted, in the terminal 24
residues of the protein.
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5 was diffusely distributed throughout the
cytoplasm (Fig. 3C), whereas GFP-BsMinD3 (Fig.
3D) and GFP-BsMinD2 (not shown) exhibited a
peripheral localization pattern that was indistinguishable from that of
GFP-BsMinD (Fig. 3A) and GFP-EcMinD
(Fig. 1C). Cells expressing GFP-BsMinD4
exhibited an intermediate phenotype in which only 10–20% of cells
showed distinct peripheral localization of the fusion protein (not
shown). This effect was not due to poor expression of the fusion
protein because Western blot analyses (not shown) indicated that the
MinD protein levels were very similar in cells expressing WT
GFP-BsMinD and each of the truncation mutants. Thus, it
seems that the MTS highlighted in Fig. 2 corresponds to the absolute
minimal region required for membrane localization of MinD, but that
optimal localization of some MinD proteins may require additional
C-terminal residues. We conclude that the C-terminal region of MinD contains a membrane-targeting sequence that is conserved at least in Gram-negative and Gram-positive bacteria, and most likely also in archaea and plastids. It is salient to note that MinD from the Gram-negative coccus Neisseria gonorrhoeae was recently shown to be functional in E. coli (19), which indicates that N. gonorrhoeae MinD must be able to localize to the E. coli inner membrane, consistent with the fact that it contains a canonical MTS (Fig. 2).
The MinD MTS Is an Amphipathic Helix. The MTS regions of MinD from archaea, eubacteria, and chloroplasts are predicted to be helical by numerous secondary structure prediction programs. When representative MinD MTSs from each domain of life are arranged on a helical wheel (Fig. 4 A–C) they form highly amphipathic helices; one face of the helix is highly hydrophobic and consists primarily of Phe, Leu, and Met residues, whereas the opposing face is polar and is generally dominated by positively charged Lys and Arg residues. Protein interactions with the lipid bilayer are commonly mediated by amphipathic helices, which are often structured only in the presence of phospholipid membranes (20, 21). We therefore wondered whether the predicted helical structure of the MTS was important for membrane localization of MinD.
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First, we constructed a mutant of EcMinD in which three helix-compatible residues (Ala-Lys-Ile) were inserted between Leu-264 and Lys-265 of the MTS; these residues are equivalent to the three extra residues in the B. subtilis MTS. This mutant (EcMinDIns3) is predicted to be helical and, furthermore, the size of the insertion ensures that the amphipathicity of the MTS is maintained (see helical wheel in Fig. 4E). GFP-EcMinDIns3 exhibited a distinct peripheral localization pattern that was indistinguishable from that of GFP-EcMinD (Fig. 4G). Next, we examined the localization of a mutant in which two helix-compatible residues (Ala-Lys) were inserted between Leu-264 and Lys-265 of the MTS; this insertion should maintain the helicity of the MTS, but it destroys the polar-apolar residue phasing so that a Lys residue is positioned on the otherwise hydrophobic face of the helix and a hydrophobic Leu residue is positioned on the polar face (see helical wheel in Fig. 4F). We found that this mutant (EcMinDIns2) was diffusely distributed throughout the cytoplasm (Fig. 4H). Insertion of two helix-breaking residues (Gly-Asn) between Leu-264 and Lys-265 of the MTS also caused aberrant cytoplasmic localization of MinD (data not shown).
We further explored whether the amphipathic nature of the MTS
helix is critical for membrane targeting by examining the localization
of an EcMinD mutant in which Leu-267, a highly conserved
residue located on the hydrophobic face of the putative MTS helix (see
Fig. 4D), was mutated to a negatively charged Glu residue.
Glu was chosen because it is compatible with helix formation, and a
Leu 
Glu mutation on the hydrophobic face of the N-terminal
amphipathic helix of sterol carrier protein-2 has been shown to abolish
its membrane-targeting function (20). As shown in Fig. 4I,
the GFP-EcMinD(L267E) mutant was found to be localized
diffusely throughout the E. coli cytoplasm. We conclude that
both the helicity and the amphipathicity of the MTS is critical for its
membrane-targeting function.
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Discussion |
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Until now, the mechanism by which MinD is recruited to the membrane has proved elusive. In this study we have shown that localization of MinD to the bacterial membrane is mediated by a highly conserved sequence in the C-terminal region of the protein that we refer to as the membrane-targeting sequence or MTS (Fig. 2). The consensus MTS is KG[FLI][LFI]X3–4KR[LFI][FL], where X3–4 is a three- or four-residue insertion found in some MinD proteins. Significant conservation occurs outside this region within Gram-negative eubacteria, but the MTS is the only portion of the C-terminal region that is conserved across phyla. The MinD MTS spans only 8–12 residues, but MTSs in several other proteins are similarly small; for example, the membrane-anchoring sequence of the E. coli signal-transducing enzyme IIAGlucose comprises only nine residues at the extreme N terminus of the protein (26), whereas a 12-residue amphipathic helix mediates membrane attachment of ADP-ribosylation factor 1 (ARF1) (27).
The observation that the extreme C-terminal region of MinD contains a MTS explains why large C-terminal fusions, such as GFP, abolish MinD function (22); we presume that these fusions sterically impede the interaction of the MinD MTS with the cytoplasmic membrane, thus abrogating the membrane association–dissociation cycle necessary for the biological function of MinD (13, 14).
The MinD MTS Is Not Present in Other Members of the ParA SuperFamily. MinD is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif (11, 12, 28). Examination of previously compiled ParA alignments (e.g., see supporting information for ref. 11, www.pnas.org/cgi/content/full/97/26/14656/DC1/3), as well as extensive BLAST searches using the MinD MTS, revealed that this motif is confined specifically to MinD and is absent from other members of the ParA superfamily. Consistent with this observation, we are not aware of any ParA superfamily proteins, apart from MinD, that specifically localize to the membrane. Several ParA proteins involved in chromosome and plasmid partitioning undergo a bipolar oscillation like MinD, but they associate with the nucleoid rather than the bacterial membrane (29–31).
The MinD MTS might be a useful criterion for distinguishing between true MinD orthologs and other ParA superfamily members in organisms that are recalcitrant to genetic manipulation. For example, the annotated genome sequence of the spirochete Borrelia burgdorferi includes three chromosomally encoded "MinD-related proteins" (YLXH-1, YLXH-2, and YLXH-3), but only one of these (YLXH-1) contains the MinD MTS signature. Similarly, P. furiosus contains three apparent MinD homologs, but only one contains the MinD MTS signature. The MinD MTS does not seem to be present in either of the Pyrococcus proteins chosen for structure determination (16, 17), and hence, these proteins may not be functional homologs of MinD. However, the MTS is present in the A. fulgidus MinD homolog used for structure determination (15), and hence, this protein is likely to be an authentic MinD ortholog.
A Model for Membrane Association of MinD. The MinD MTSs are predicted to be helical and, when displayed on a helical wheel, the putative helices are highly amphipathic. One face of the helix is composed entirely of hydrophobic residues (most often Phe and Leu), whereas the opposing polar face usually contains several positively charged Arg and Lys residues and is generally devoid of anionic residues (Fig. 4 A–D). Experiments with several insertion mutants (Fig. 4 E–H) and an EcMinD(L267E) point mutant (Fig. 4I) suggested that both the helicity and amphipathicity of the MinD MTS are important for its membrane-targeting function.
Amphipathic helices mediate the association of numerous proteins with biological membranes (32, 33). These helices are generally unstructured in the absence of membrane (20, 21, 26, 34) and, in most cases that have been examined experimentally, they preferentially associate with anionic phospholipids because of the large number of positively charged residues on the polar face of the helix; examples include sterol carrier protein-2 (20), CTP:phosphocholine cytidyltransferase (34), and the mammalian GTPase RGS4 (21). The marked preponderance of cationic residues on the polar face of the MinD MTS suggests that this motif most likely mediates the interaction of MinD with biological membranes by preferentially interacting with anionic phospholipids. Consistent with this hypothesis, it was previously shown that mutation of Gly-262 on the polar (cationic) surface of the EcMinD MTS to a negatively charged Asp residue results in a minicelling phenotype (35). The cellular localization of the G262D mutant has not been examined, but we predict that the minicelling phenotype is a direct result of impaired membrane association of EcMinD.
Amphipathic helices that contain a large number of positively charged residues on their polar face usually align parallel to the membrane surface, because it is thermodynamically highly unfavorable to insert Lys and Arg residues into the hydrophobic interior of the bilayer (33). We anticipate that this is likely to be the case for the MinD MTS. As shown in Fig. 5, the helix orientation is likely to be such that the hydrophobic residues interact directly with lipid acyl chains, whereas the cationic residues on the opposite face of the helix interact with the headgroups of anionic phospholipids (21, 33). This type of superficial protein–lipid interaction is often used by proteins whose activity is regulated by reversible membrane association; for example, the amphipathic membrane-targeting helix of CTP:phosphocholine cytidyltransferase mediates interconversion between the inactive cytoplasmic and active membrane-bound forms of the enzyme (34).
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What Regulates MTS Association–Dissociation Cycles? We presume that MinE-stimulated ATP hydrolysis leads to detachment of the MinD MTS from the membrane, which raises two critical questions: What is the molecular basis of MTS detachment from the membrane, and what prevents it from immediately reassociating with the bilayer? We suggest two possibilities. First, it is possible that MinE interacts directly with the MTS, thereby causing it to detach from the membrane and remain cytosolic so long as it continues to associate with MinE. Second, it is possible that ATP hydrolysis provokes a change in the conformation and/or polymerization state of MinD that makes it less favorable for the MTS to interact with the bilayer. One possible scenario is that, after detachment from the membrane, the MTS associates with the core of the MinD protein such that the hydrophobic face of the helix becomes buried. This mechanism is used by several GTPases to modulate their interaction with biological membranes. For example, the Ras-related GTPase ARF1 associates reversibly with membranes in a GTP-dependent manner and this interaction is mediated by an N-terminal amphipathic helix (27). In the crystal structure of the GDP-bound form of ARF1 (36), the N-terminal helix is tightly associated with the rest of the protein, with residues on the hydrophobic face directed toward the protein core. However, GTP binding provokes a conformational change in ARF1 that exposes these hydrophobic residues and allows them to interact with membrane lipids (27, 37).
Only minor differences exist between the structures of a
presumptive P. furiosus MinD homolog (16) complexed with ADP
and the nonhydrolyzable ATP analog adenosine
5'-[,
-methylene]triphosphate (AMPPCP), which suggests that
membrane association of the MinD MTS is not controlled by an
ATP-induced conformational switch. However, AMPPCP may not be a good
ATP analog in the case of MinD because ATP and adenosine
5'-O-(3-thiotriphosphate) (ATPS) promote interaction of
MinD with phospholipid vesicles, whereas AMPPCP does not (14).
Furthermore, the P. furiosus protein may not be a true MinD
ortholog because it lacks a canonical MinD MTS, and therefore its
crystal structure may not be indicative of the conformational changes
that occur when MinD binds ATP. Consequently, it remains possible that
ATP binding induces a conformational change in MinD that promotes
membrane attachment of the MTS. Structures of an authentic MinD
complexed with ADP and ATPS should help to resolve this issue.
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Acknowledgements |
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minCDE strain. This work was
supported by National Institutes of Health Grants GM-48583 (to G.F.K.)
and GM-60632 (to L.I.R.). |
Footnotes |
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To whom correspondence should be addressed. E-mail:
glenn{at}psel.uchc.edu. 
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) and
His6-EcMinD
). MRE is the mean residue ellipticity in
degrees•cm2•dmol–1.
