Kinase Associated-1 Domains Drive
MARK/PAR1 Kinases to Membrane Targets
by Binding Acidic Phospholipids
Katarina Moravcevic,1,2Jeannine M. Mendrola,1Karl R. Schmitz,2Yu-Hsiu Wang,4,5David Slochower,2,5
Paul A. Janmey,2,3,5and Mark A. Lemmon1,2,*
1Department of Biochemistry and Biophysics
2Graduate Group in Biochemistry and Molecular Biophysics
3Department of Physiology
University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
4Department of Chemistry
5Institute for Medicine and Engineering
University of Pennsylvania, Philadelphia, PA 19104, USA
Phospholipid-binding modules such as PH, C1, and
C2 domains play crucial roles in location-dependent
regulation of many protein kinases. Here, we identify
the KA1 domain (kinase associated-1 domain), found
at the C terminus of yeast septin-associated kinases
(Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1
kinases, as a membrane association domain that
binds acidic phospholipids. Membrane localization
of isolated KA1 domains depends on phosphatidyl-
serine. Using X-ray crystallography, we identified a
structurally conserved binding site for anionic phos-
pholipids in KA1 domains from Kcc4p and MARK1.
Mutating this site impairs membrane association of
both KA1 domains and intact proteins and reveals
the importance of phosphatidylserine for bud neck
localization of yeast Kcc4p. Our data suggest that
KA1 domains contribute to ‘‘coincidence detection,’’
allowing kinases to bind other regulators (such as
septins) only at the membrane surface. These find-
ings have important implications for understanding
MARK/PAR1 kinases, which are implicated in Alz-
heimer’s disease, cancer, and autism.
Regulation of cellular processes requires precisely controlled
intermolecular interactions that alter the location and/or activity
of effector proteins (Scott and Pawson, 2009), typically driven
by protein modules that recognize specific features of proteins,
nucleic acids, or membranes (Seet et al., 2006). Several protein
modules recognize anionic membrane phospholipids, including
PH, C2, PX, and FYVE domains (Lemmon, 2008). Some recog-
are tightly regulated. Others bind phosphatidylserine (PtdSer),
which is concentrated in the plasma membrane inner leaflet
(Yeung et al., 2008) and constitutes approximately 20% of phos-
pholipid (Stace and Ktistakis, 2006).
Many more cellular functions appear to depend on anionic
phospholipids than can be explained by currently understood
phospholipid-binding domains (Audhya et al., 2004; Halstead
et al., 2005; McLaughlin and Murray, 2005; Yu et al., 2004).
Indeed, in a microarray-based analysis of the expressed S. cer-
evisiae proteome, over 100 proteins that contain no known lipid-
2001). Here, we describe an analysis of the membrane associa-
tion properties of these yeast proteins, from which we have iden-
tified several additional potential phospholipid-binding domains.
We focus in this report on a membrane-targeting domain found
at the C terminus of the S. cerevisiae septin-associated protein
kinases Kcc4p, Gin4p, and Hsl1p. These kinases are involved
in septin organization or in the yeast morphogenesis checkpoint
that coordinates cell-cycle progression with bud formation (Lew,
2003; Longtine and Bi, 2003; Shulewitz et al., 1999). They
become activated at the bud neck and are involved in septin
ring assembly and/or promote Swe1p degradation to allow
entry into mitosis (Barral et al., 1999; Sakchaisri et al., 2004).
The C-terminal phospholipid-binding domain of the septin-asso-
ciated kinases is required for their bud neck localization and
function and appears to bind phosphatidylserine in vivo. Using
X-ray crystallography, we found that this phospholipid-binding
domain has the same fold as the KA1, or kinase associated-1
domain (Pfam accession PF02149), one of the only common
domains in protein kinases to which no function has yet been
ascribed (Manning et al., 2002; Tochio et al., 2006).
KA1 domains are also found at the C termini of mammalian
Ser/Thr kinases that phosphorylate microtubule-associating
proteins (MAPs) such as tau, promoting their detachment from
microtubules and thus reducing microtubule stability (Drewes
et al., 1997). These kinases comprise the MARK/PAR1 family,
which includes MAP/microtubule affinity-regulating kinase
966 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
(MARK) and partitioning-defective 1 or PAR1 (Matenia and Man-
delkow, 2009; Timm et al., 2008), as well as the S. cerevisiae
Kin1/2 kinases (Tassan and Le Goff, 2004). MARK/PAR1 kinases
are related to the AMP-activated protein kinase (AMPK)/Snf1
family (Manning et al., 2002; Marx et al., 2010). They are
frequently found associated with membrane structures and
participate in diverse processes from control of the cell cycle
and polarity to intracellular signaling and microtubule stability
(Marx et al., 2010; Tassan and Le Goff, 2004). MARK/PAR1
kinases have been implicated in carcinomas, Alzheimer’s dis-
ease (through tau hyperphosphorylation), and autism (Gray
et al., 2005; Hurov et al., 2007; Maussion et al., 2008; Timm
and human kinases bind anionic phospholipids, thus ascribing
a function to this poorly understood domain and providing
important clues as to how activation of these AMPK-related
Screen for Unidentified Phospholipid-Binding Domains
Zhu et al. (2001) reported phosphoinositide binding for 128 of
5800 protein products from S. cerevisiae open reading frames
(ORFs) arrayed on proteome chips—excluding dubious ORFs
and integral membrane proteins. We selected 62 of these for
further analysis (15 of which were protein kinases), including all
‘‘strong binders’’ defined by Zhu et al. (2001) plus potentially
interesting ‘‘weak binders.’’ We first tested in vivo membrane
association of these 62 proteins using an S. cerevisiae Ras
rescue assay (Isakoff et al., 1998; Yu et al., 2004). Each protein
was fused to constitutively active (Q61L), nonfarnesylated,
Ha-Ras and expressed in cdc25tsyeast cells—which harbor
a temperature-sensitive mutation in the Ras guanine nucleotide
exchange factor Cdc25p. If the test protein drives plasma mem-
brane recruitment of this Ha-Ras fusion, it promotes growth
above the restrictive temperature (complementing the cdc25ts
allele) by overcoming the block in endogenous Ras activation
(Isakoff et al., 1998). Of the 62 proteins analyzed, 33 promoted
membrane recruitment of constitutively active Ha-Ras (Fig-
ure S1A and Table S1A available online), consistent with them
harboring a phospholipid-binding domain. In qualitative lipid
proteins also interacted in vitro with filter-bound anionic phos-
pholipids (Table S1A), displaying a broad range of specificities.
Several of the candidate Ras rescue-positive proteins also
showed punctate or plasma membrane fluorescence when
expressed as GFP fusion proteins in yeast or HeLa cells
(Table S1A). For five of the candidate proteins (Cam1p, Dps1p,
Kcc4p, Rgd1p, and Stp22p), Ras rescue analysis of deletion
targeting (Table S1B). We focus here on Kcc4p.
A Membrane-Targeting Domain at the C Terminus
of the Septin-Associated Kinase Kcc4p
In studies of the septin-associated kinase Kcc4p, Ras rescue
analysis identified a C-terminal 160 aa fragment (aa 877–1037)
that is sufficient to drive Ha-Ras membrane recruitment in yeast
cells (Figure 1A). This fragment also displays strong plasma
membrane association when overexpressed as a GFP fusion
protein in either S. cerevisiae or human HeLa cells (Figure 1B),
suggesting recognition of a lipid that is common to yeast and
target at the membrane.
The Kcc4p C-Terminal Domain Binds
As shown in Figure 1C, purified protein corresponding to resi-
dues 901–1037 from the Kcc4p C terminus (Kcc4p901-1037) binds
‘‘promiscuously’’ to PtdIns(4,5)P2and other acidic phospho-
lipids in surface plasmon resonance (SPR) studies. Overlay
studies of intact Kcc4p (Table S1A) showed a similar lack of
specificity, consistent with the binding to several phosphoinosi-
tides reported previously by Zhu et al. (2001). Kcc4p901–1037
bound with similar affinities to membranes containing 10%
(mole/mole) PtdIns(4,5)P2, 20% (mole/mole) phosphatidic acid
(PA), or 20% (mole/mole) PtdSer—all in a dioleoylphosphatidyl-
choline (DOPC) background. The binding data fit well to simple
hyperbolic curves with apparent dissociation constant (KD)
values from 3–10 mM (Table S2), in the same range reported for
several other phospholipid-interaction
2008). The amount of Kcc4p901–1037bound at saturation (Bmax)
scaled with anionic phospholipid content for PtdIns(4,5)P2or
PtdSer (Figure 1D). Interestingly, in all studies, Bmaxwas propor-
tional to the anticipated negative charge density on the SPR
sensorchip surfaces (rather than number of lipid molecules),
assuming charge valences of ?4, ?2, and ?1 for PtdIns(4,5)P2,
PA, and PtdSer, respectively, at pH 7.4 (McLaughlin and Murray,
2005). As shown in Figure 1C, Bmaxwas approximately 2000
resonance units (RUs) for membranes containing either 10%
PtdIns(4,5)P2(charge ?4) or 20% PA (charge ?2) and approxi-
1:1 complexes, binding stoichiometry depends on lipid charge ?
each Kcc4p901–1037chain binding four times more PtdSer mole-
cules (charge ?1) than PtdIns(4,5)P2molecules (charge ?4).
We also used a centrifugation-based sedimentation assay to
analyze Kcc4p901–1037binding to small unilamellar vesicles (Kav-
ran et al., 1998). Only background levels of Kcc4p901–1037sedi-
mented with vesicles with no net charge, i.e., those containing
100% phosphatidylcholine (PC) or 20% (mole/mole) phosphati-
dylethanolamine (PE) in a PC background (Figure 1E). By con-
trast, vesicles containing 20% (mole/mole) of the anionic phos-
pholipids PtdSer or PtdIns sedimented the majority of the
Kcc4p901–1037when anionic lipid was present at R50 mM. Diva-
lent cations did not significantly alter the affinity or specificity of
phospholipid binding by Kcc4p901–1037. Neither elevating diva-
lent cation levels (by adding 10 mM CaCl2and 1 mM MgCl2)
nor depleting them (by adding 1 mM EDTA) changed apparent
KDvalues by more than 2-fold (Table S2).
Related C-Terminal Domains in Gin4p and Hsl1p Septin-
Associated Kinases Also Bind Anionic Phospholipids
to the SMART, Pfam, and UniProt databases is the N-terminal
kinase domain (Figure 1A). However, BLAST searches (Altschul
et al., 1990) identify an ?130 amino acid region related to
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. 967
Kcc4p901–1037at the C termini of the functionally related S. cere-
visiae kinases Gin4p and Hsl1p (Figure 2A and Figure S2). The
Gin4p C terminus (residues 1007–1142) shares 41% sequence
identity with Kcc4p901–1037, and the Hsl1p C terminus (residues
1379–1518) is more distantly related (sharing just 16% identity
with Kcc4p901–1037). As shown in Figure 2B, fusing these
C-terminal regions from Gin4p or Hsl1p to Q61L Ha-Ras allowed
complementation of the cdc25tsallele in Ras rescue assays. The
Phosphatidic acid (20%)
Max Binding (RUs)
Total Available Lipid (μM)
Figure 1. A C-Terminal Domain in Kcc4p
Binds Phospholipids and Associates with
(A) A C-terminal 160 aa Kcc4p fragment (residues
877–1037) is necessary and sufficient for mem-
rescuing 37?C growth of cdc25tsyeast cells. Serial
dilutions of yeast cultures expressing each Kcc4p
fragment were spotted in duplicate onto selection
plates and incubated at 25?C or 37?C.
(B) The same C-terminal Kcc4p fragment, fused to
GFP, shows plasma membrane localization in
S. cerevisiae and HeLa cells.
(C) SPR studies of Kcc4p901–1037binding to DOPC
membranes containing 10% (mole/mole) PtdIns
(4,5)P2(KD= 10.6 ± 1.1 mM), 20% (mole/mole)
phosphatidic acid (KD= 10.2 ± 0.3 mM), or 20%
(mole/mole) PtdSer (KD= 7.8 ± 3.4 mM). Binding
dent experiments, and mean KDvalues ± standard
deviation are quoted (Table S2).
(D) SPR signals at saturation show that maximal
Kcc4p901–1037 binding scales with the negative
charge density in immobilized membranes. Mean
Bmaxvalues ± standard deviations (for >3 experi-
ments) are plotted for membranes containing the
noted percentages (mole/mole) of PtdIns(4,5)P2
(valence ?4 at pH 7.4) and PtdSer (valence ?1 at
(E) In vesicle sedimentation studies, His6-Kcc4p901–1037(at 50 mM) binds small unilamellar vesicles containing 20% (mole/mole) phosphatidylinositol (PtdIns) or
20% (mole/mole) PtdSer in a brominated PC background, but not to phosphatidylethanolamine (PE). At 500 mM ‘‘total available lipid,’’ 100 mM of PtdIns, PE, or
PtdSer is available for binding on the vesicle outer leaflet. Mean ± standard deviation is plotted for at least three independent experiments.
Figure S1 and Tables S1A and S1B summarize results for other potential phosphoinositide-binding proteins.
Figure 2. The Membrane-Targeting Domain
of Kcc4p Is Conserved in Gin4p and Hsl1p
(A) Alignment of C-terminal fragments from the
three S. cerevisiae septin-associated kinases
Kcc4p, Gin4p, and Hsl1p. Acidic residues are
red, basic blue, hydrophobic green, and hydro-
philic plum. Colored blocks or text denote posi-
tions at which two or more residues are identical
or similar, respectively. See also Figure S2.
(B) Ras Rescue studies of Gin4p943–1142 and
(C) GFP/Gin4p1003–1142 and GFP/Hsl1p1358–1518
localize to the plasma membrane in S. cerevisiae
(D) SPR studies show that GST/Gin4p943–1142
binds DOPC membranes containing 20% (mole/
mole) phosphatidic acid (KD = 5.7 ± 0.5 mM),
20% PtdSer (KD= 8.6 ± 2.6 mM), or 10% PtdIns
(4,5)P2(KD= 4.7 ± 0.3 mM). Binding curves are
iments. Note that GST dimerization causes over-
estimation of apparent binding affinity in this assay
(Yu et al., 2004).
968 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
Gin4p and Hsl1p C termini also showed robust plasma mem-
brane localization when expressed in yeast cells as GFP fusion
proteins (Figure 2C). Moreover, the Gin4p C-terminal domain
(expressed in E. coli as a GST fusion protein) bound PA, PtdIns
(4,5)P2, and PtdSer in SPR studies (Figure 2D), resembling
the in vitro interactions seen for Kcc4p901–1037(although with
different charge dependence, interpretation of which is compli-
cated by dimerization of the fused GST). The Gin4p and Hsl1p
C termini therefore have broadly similar membrane-binding
out that Gin4p and Hsl1p were not found in the proteome-wide
screen of yeast phospholipid-binding proteins described by
Zhu et al. (2001), arguing that additional, as-yet-unidentified,
S. cerevisiae phospholipid-binding proteins may exist.
Loss of Phosphatidylserine Impairs Membrane
Targeting of Kcc4p, Gin4p, and Hsl1p C-Terminal
To determine which cellular phospholipids are important for
in vivo membrane association of the C termini from Kcc4p,
Gin4p, and Hsl1p, we assessed their localization (as GFP fusion
proteins) in S. cerevisiae mutants harboring specific phospho-
lipid synthesis defects. Plasma membrane localization was not
detectably altered when levels of PtdIns(4,5)P2 or PtdIns4P
were reduced by manipulation of temperature-sensitive yeast
strains (Stefan et al., 2002), arguing that neither of these phos-
phoinositides plays a dominant role (Figure S3). By contrast, in
cho1D cells that lack PtdSer (Hikiji et al., 1988), the degree
of plasma membrane association of each domain was reduced
significantly (Figure 3). Ratios of plasma membrane to cyto-
solic fluorescence (FPM/FCyt: see Experimental Procedures)
in wild-type cells were 1.4 ± 0.35, 1.5 ± 0.08, and 2.9 ± 1.0,
respectively, for GFP/Kcc4p877–1037, GFP/Gin4p1003–1142, and
GFP/Hsl1p1358–1518,similar to the FPM/FCytratio of 1.5 ± 0.16
measured for the lactadherin discoidin-type C2 domain previ-
ously characterized as a specific PtdSer probe (Yeung et al.,
2008). Loss of PtdSer in cho1D cells reduced FPM/FCytratios to
0.53 ± 0.15 (Kcc4p877–1037), 0.93 ± 0.20 (Gin4p1003–1142), and
Figure 3. Phosphatidylserine Depletion Reduces
Gin4p1003–1142, and Hsl1p1358–1518
Localization of GFP-fused Kcc4p877–1037, Gin4p1003–1142,
and Hsl1p1358–1518in wild-type yeast cells (left) and in
cho1D cells, which lack PtdSer. The lactadherin C2
domain was used as a control probe for PtdSer (Yeung
et al., 2008). The five panels shown for each GFP fusion
in cho1D cells reflect the range of localization phenotypes
observed,illustrating reduced plasma membrane associa-
tion. Figure S3 shows that reducing phosphoinositide
levels has no such effect.
0.95 ± 0.13 (Hsl1p1358–1518)—mirroring the
effect on the PtdSer-specific lactadherin C2
domain (FPM/FCyt= 0.61 ± 0.20).
surface-potential probes and the lactadherin
C2 domain have shown that the plasma mem-
brane inner leaflet is the most negatively charged of cyto-
plasmic-facing membranes, and that PtdSer is the primary
determinant of this surface charge (Yeung et al., 2006, 2008).
C-terminal domains from the septin-associated kinases appear
to resemble these nonspecific surface-potential probes. They
show preferential targeting to the plasma membrane that is
dependent on PtdSer, although they do not specifically recog-
nize this lipid. The residual plasma membrane association seen
in cho1D cells for these domains (Figure 3) may reflect their
ability to bind either PtdIns (see Figure 1E), levels of which are
known to be elevated in cho1D cells (Hikiji et al., 1988), or other
less abundant anionic plasma membrane phospholipids.
Structure of the Kcc4p C-Terminal Domain Reveals
a KA1 Domain Fold
In an effort to understand anionic phospholipid binding by
C-terminal domains from the septin-associated kinases, we
determined the X-ray crystal structure of Kcc4p917–1037to 1.7 A˚
resolution (see Table S3). The domain contains two interacting
a helices (a1 and a2) that lie on the concave surface of a five-
stranded antiparallel b sheet (Figure 4A and Figure S4). A short
b strand (b1) precedes helix a1, which is then followed by
a four-stranded b-meander (b2–b5) and a C-terminal a helix
(a2). Remarkably, the structure of Kcc4p917–1037is very similar
to that of the extended KA1 domain from the MARK3 human
MAP/microtubule affinity-regulating kinase (Tochio et al.,
2006), depicted in Figure 4B (Protein Data Bank [PDB] ID
1UL7). KA1 domains were initially defined as a Pfam domain
family of ?50 amino acids (PF02149) at the C termini of kinases
from the MARK/PAR1/Kin family (Matenia and Mandelkow,
2009; Tassan and Le Goff, 2004; Timm et al., 2008). NMR
structural studies (Tochio et al., 2006) showed that the stable
KA1 domain in MARK3 actually contains ?100 amino acids.
The 118 residue phospholipid-binding domain at the Kcc4p
C terminus that we have identified here also appears to be
a KA1 domain. It contains all secondary structure elements
seen in MARK3-KA1, plus a short additional a helix at its amino
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. 969
Kcc4p and MARK3 KA1 domains overlay very well (with Ca posi-
tion root-mean-square [rms] deviation of just 2.4 A˚), despite
sharing only 10% sequence identity—explaining the failure to
identify this domain through sequence analysis. A structure-
based sequence alignment of KA1 domains from the MARK/
PAR1/Kin family and the Kcc4p/Gin4p/Hsl1p kinases is shown
in Figure S2.
KA1 Domains from Human MARK/PAR1 Kinases Bind
Although speculated to participate in autoregulatory intramo-
lecular interactions in MARK/PAR1 kinases (Marx et al.,
2010), no clear function has been ascribed to KA1 domains.
Having identified the Kcc4p KA1 domain as a phospholipid-
binding domain, we next asked whether previously recognized
KA1 domains from human MARK1, MARK3, and MELK
(maternal embryonic leucine zipper kinase) also associate
with cell membranes and bind phospholipids. As shown in Fig-
ure 5A, all of these KA1 domains recruit Q61L Ha-Ras fusions
to yeast cell membranes, complementing the cdc25tsmutation
in Ras rescue assays. GFP fusions of the MARK1 and MARK3
KA1 domains showed substantial plasma membrane localiza-
tion in HeLa cells (Figure 5B). Moreover, the MARK1, MARK3,
and MELK KA1 domains (as GFP fusions) showed robust
plasma membrane localization in S. cerevisiae, with FPM/FCyt
ratios ranging from 1.8 to 3.1 (Figure 5C). Again, these values
were reduced by ?50% in PtdSer-deficient cho1D cells
(Figure 5C) but were not significantly altered in mutant yeast
strains with reduced phosphoinositide levels (Figure S5). The
subcellular localization properties of KA1 domains from human
MARK1, MARK3, and MELK therefore appear similar to those
seen for the Kcc4p, Gin4p, and Hsl1p KA1 domains identified
here. In addition, purified monomeric MARK1-KA1 showed
essentially the same in vitro phospholipid-binding characteris-
tics as Kcc4p-KA1, binding to vesicles that contain PtdSer,
PA, or PtdIns(4,5)P2(Figure 5D) with KDvalues in the 2.3 mM–
8.9 mM range (Table S2), and with Bmaxvalues that scale with
membrane charge density. The KA1 domains from MARK/
PAR1 family kinases thus appear to be phospholipid-binding
domains that are likely to promote membrane association of
their host proteins in cells. Indeed, Alessi and colleagues (Go ¨r-
ansson et al., 2006) previously implicated the KA1 domain as
an important membrane localization determinant in MARK3
mutants that fail to bind 14-3-3 proteins. Our findings suggest
that this observation reflects MARK3-KA1 binding to acidic
phospholipids and argue that the KA1 domain should be
Figure 4. The Kcc4p C Terminus Adopts a KA1 Domain Fold
(A) Cartoon representation of Kcc4p917–1037structure. Helices aN, a1, and a2 are marked, as are strands b1–b5. Two orthogonal views are shown. See also Fig-
(B) NMR structure (Tochio et al., 2006) of the KA1 domain from mouse MARK3 (PDB ID 1UL7), in the same orientations used in (A) for Kcc4p917–1037.
(C) Ca overlay of MARK3-KA1 (cyan) with Kcc4p917–1037(magenta). The N-terminal part of Kcc4p917–1037, including helix aN, was removed for clarity.
970 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
considered as a bona fide membrane-targeting/anionic phos-
Basic Regions on the KA1 Domain Surface Drive
To understand how KA1 domains interact with negatively
charged membranes, we analyzed features common to the
structure of the yeast Kcc4p KA1 domain and a crystal structure
of the human MARK1 KA1 domain that we determined to 1.7 A˚
resolution (see Table S3). Both have notable positively charged
patches and/or crevices on their surfaces (Figure 6) that result
from basic side-chain arrangements reminiscent of headgroup-
binding sites in other phospholipid-interaction domains (Hurley,
2006; Lemmon, 2008).
For Kcc4p-KA1, clear electron density could be seen for two
bound sulfate ions, 27 A˚apart, which lie on either side of a posi-
tively charged region that stretches across the width of the
domain in the orientation shown in Figure 6A and encircles the
b3/b4 loop that projects prominently from its surface. One of
these sulfates (SO4#1) interacts primarily with lysine side chains
in the aN/b1 loop (K932) and b5 (K1010), and it lies close to
K1016 in the amino-terminal part of helix a2 (Figure 6A and
Figure S4A). Adjacent electron density (?3 A˚away) is best fit
with a glycerol molecule that contacts K1010 in strand b5 plus
serine and threonine side chains (S1014 and T1015) at the
beginning of helix a2 (Figure S4A). Intriguingly, in a second
crystal form (Table S3) density for a tartrate ion replaces both
SO4#1 and the bound glycerol (Figure S4B), implicating this
region as an important anion binding site in Kcc4p-KA1. The
second sulfate in Figure 6A (SO4#2) lies in a basic pocket on
the Kcc4p-KA1 surface formed largely by side chains from the
helix a1 C terminus (K959) and the a1/b2 loop (K964).
The locations of bound anions in crystal structures of
membrane-targeting domains frequently reveal the binding sites
for phospholipid headgroups (Hurley, 2006; Lemmon, 2008;
Wood et al., 2009). We therefore used mutagenesis to investi-
gate the importance of the SO4#1 and SO4#2 binding sites for
in vivo membrane association of Kcc4p-KA1. When pairs of
basic residues were mutated (Figure 6A), plasma membrane
localization of GFP/Kcc4p-KA1 was only impaired when one or
both mutated residues contributed to binding of one of these
sulfates (K932, K1007, K1010, K1016, K1020, K964, and K978
were implicated). Importantly, mutations at both sulfate-binding
Phosphatidic acid (20%)
Figure 5. KA1 Domains from Human MARK/PAR1 Kinases Bind Phospholipids
(A) KA1domains from human MARK1 (aa 648–795),human MARK3 (aa 589–729),and human MELK (aa500–651)all drive membrane recruitment of Ha-RasQ61L
fusions in Ras rescue studies.
(B) GFP-fused human MARK1 and MARK3 KA1 domains show plasma membrane localization in HeLa cells. Unexplained nuclear localization of the MELK-KA1
fusion made interpretation of its behavior difficult (not shown).
(C) GFP-fused KA1 domains from human MARK1, MARK3, and MELK show robust plasma membrane localization in S. cerevisiae cells, which is diminished in
cho1D cells that lack PtdSer. Mean FPM/FCytratios for each experiment (±standard deviation) are quoted in individual panels. Figure S5 shows that manipulating
phosphoinositide levels in yeast cells does not affect membrane targeting of MARK family KA1 domains.
(D) Purified MARK1-KA1 binds membranes containing phosphatidic acid (20%), PtdSer (20%), or PtdIns(4,5)P2(10%) in SPR studies. Binding curves are repre-
sentative of at least three independent experiments. Mean apparent KDvalues (±standard deviation) are listed in Table S2.
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. 971
sites diminished membrane recruitment, suggesting that the
KA1 domain makes multiple contacts with the bilayer surface.
Engaging both the SO4#1 and SO4#2 sites in binding to a
membrane surface is difficult to envision without the b3/b4
loop penetrating the bilayer. This loop contains several hydro-
phobic side chains (with sequence VNDSILFL) and resembles
‘‘membrane insertion loops’’ reported in C2, PX, and FYVE
domains (Cho and Stahelin, 2005; Lemmon, 2008). As shown
lipid-containing monolayers that have packing densities similar
to those estimated for cell membranes (Demel, 1994; Marsh,
1996)—resembling C2, PX, FYVE, and some PH domains in
this respect (Stahelin et al., 2007).
Only the SO4#1/glycerol-binding site of Kcc4p-KA1 is
conserved in the hMARK1 KA1 domain—in location, charge
characteristics (Figure 6B), and sequence (Figure S2). It lies in
the most sequence-conserved region of aligned KA1 domains
that encompasses strand b5, helix a2, and the loop that
3/ 4 loop
3/ 4 loop
Figure 6. Potential Phospholipid-Binding
Sites on Kcc4p and MARK1 KA1 Domains
(A) Kcc4p-KA1 is shown in surface representation
(left: with electrostatic surface potential—blue,
positive; red, negative) and in cartoon form (right:
same orientation). The two ordered sulfate ions
(SO4#1 and SO4#2) and the glycerol molecule
close to SO4#1 are marked, as is the b3/b4 loop.
Figure S4 shows the tartrate ion that replaces
SO4#1 and the glycerol in another crystal form.
Noted residues were mutated in pairs to serine,
expression confirmed by western blotting (not
shown), and effects on plasma membrane locali-
zation of GFP fusions assessed in wild-type yeast
cells (right). Double mutations marked with red
asterisks showed significantly reduced FPM/FCyt
(mean FPM/FCyt= 1.7 ± 0.3). FPM/FCytvalues for
mutated variants were 0.81 ± 0.09 (K930S/
K932S), 0.74 ± 0.03 (K959S/K964S), 0.80 ± 0.15
(K964S/K978S), 0.92 ± 0.14 (K1007S/K1010S),
0.96 ± 0.06 (K1016S/K1020S). Residues impli-
cated in membrane binding are colored black,
whereas those at which mutations did not influ-
ence targeting are gray.
(B) Crystal structure of human MARK1-KA1 (Table
S3), shown in the same orientation as in (A).
Compared with an FPM/FCytratio of 2.0 ± 0.4 for
wild-type MARK1-KA1, mutated variants denoted
by red asterisks gave FPM/FCytvalues of 0.90 ±
describes effects of these mutations on in vitro
connects them. In addition to conserved
positive charge in this region (in b5),
all KA1 domains have serine and/or
threonine residues at the beginning of
helix a2 that contact bound glycerol
in Kcc4p-KA1 (Figure S4A) and may
interact similarly with the glycerol back-
bone of bound phospholipids. As anticipated from these obser-
vations, hMARK1-KA1 mutations in the basic patch correspond-
ing to the Kcc4p SO4#1 binding site impaired both plasma
membrane association (Figure 6B) and in vitro binding to anionic
phospholipids (Figure S6B). K773 and R774 in strand b5 of
hMARK1-KA1 appear to be important for membrane associa-
tion. Moreover, an R698S/R701S double mutation close to the
hMARK1-KA1 N terminus prevented plasma membrane associ-
ation and vesicle binding, suggesting that the basic patch
extending to the bottom left of hMARK1-KA1 in Figure 6B makes
additional contributions—perhaps functionally replacing the
SO4#2 binding site of Kcc4p-KA1. Thus, membrane association
of both the Kcc4p and the MARK1 KA1 domains appears to
involve cooperation of more than one positively charged binding
region—centered on the conserved SO4#1 binding site seen in
Kcc4p-KA1. Similar utilization of multiple binding sites has previ-
ously been described for annexins, as well as PKC-type C2, PX,
and PH domains (Lemmon, 2008).
972 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
PtdSer-Dependent Bud Neck Localization of KA1
Double mutations (K1007S/K1010S or K1016S/K1020S) that
abolish membrane localization of isolated Kcc4p-KA1 (shown
in Figure 6A) did not prevent intact Kcc4p from being targeted
to the bud neck when overexpressed in wild-type yeast cells
(Figure 7A). However, background cytoplasmic fluorescence
was increased to some extent, and simultaneous introduction
of all four KA1 domain mutations into intact Kcc4p abolished
its targeting to bud necks.
Hypothesizing that the KA1 domain must cooperate with
other domains in targeting intact Kcc4p specifically to bud
necks, we surmised that residual low-affinity PtdSer binding
by K1007S/K1010S- or K1016S/K1020S-mutated KA1 domains
might be sufficient to drive normal Kcc4p targeting in this
overexpression study. We therefore re-examined localization
of the intact GFP/Kcc4p variants in cells lacking PtdSer. As
suspected, PtdSer loss (in cho1D cells) completely abrogated
bud neck localization of K1007S/K1010S-mutated GFP/Kcc4p
(Figure 7A: see also Figure S7A). In other words, K1007S/
K1010S-mutated Kcc4p is dependent on normal plasma
membrane PtdSer levels for its targeting to the bud neck, impli-
cating PtdSer as an important determinant of Kcc4p localiza-
tion. Bud neck localization was still seen for wild-type and
K1016S/K1020S-mutated GFP/Kcc4p in cho1D cells (although
cytosolic fluorescence was increased)—suggesting that the
elevated PtdIns levels found in these cells (Hikiji et al., 1988)
may be sufficient. Western blotting confirmed that all GFP/
Kcc4p variants were expressed at or above wild-type levels
(Figure S7B). Taken together, these data show that bud neck
targeting of intact GFP/Kcc4p can be abolished either by
mutating basic residues in the KA1 domain’s anionic phospho-
lipid-binding site or—importantly—by simultaneously reducing
anionic phospholipid levels in the plasma membrane inner
leaflet and mutating the KA1 domain.
The lack of a clear phenotype for KCC4 mutations (Longtine
et al., 2000) prevented us from being able to assess functional
consequences of the KA1 domain mutations described above.
However, studies of Gin4p demonstrated a functional require-
ment for the KA1 domain (Figure 7B). Deleting the GIN4 (or
HSL1, but not KCC4) gene in S. cerevisiae leads to an elongated
bud phenotype characteristic of a G2/M delay due to morpho-
genesis checkpoint failure (Longtine et al., 1998). In gin4D cells,
this elongated bud phenotype can be rescued by overexpress-
ing a wild-type Gin4p GFP fusion (Figure 7B), and the protein is
found at bud necks. However, when just the KA1 domain (but
fails to rescue gin4D cells and is diffusely localized (Figure 7B) in
much the same way as GFP/Kcc4p harboring multiple KA1
Figure 7. Role of the KA1 Domain in Kcc4p and Gin4p
(A) Localization of wild-type and KA1 domain-mutated intact GFP/Kcc4p in wild-type yeast cells (normal) and PtdSer-deficient cho1D cells. Additional images
and western blot confirmation of intact protein expression are shown in Figure S7.
(B) Yeast cells lacking Gin4p (gin4D) show an elongated bud phenotype (left). Overexpressed GFP-fused full-length Gin4p in gin4D cells rescues this aberrant
elongated-bud morphology and is found at the bud neck in all cells. By contrast, GFP/Gin4pDKA1 fails to rescue the gin4D phenotype and remains diffuse in the
cytoplasm. Examining at least 200 cells in several experiments, the elongated phenotype was seen in 69% of gin4D cells expressing GFP alone, 78% expressing
GFP/Gin4pDKA1, and just 39% of those expressing GFP/Gin4p.
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. 973
Our search for previously undescribed phosphoinositide/phos-
pholipid-binding domains identified a small C-terminal domain
in S. cerevisiae septin-associated kinases that binds acidic
phospholipids. Crystallographic studies revealed that this is
a KA1 domain, a module previously identified at the C termini
of kinases from the mammalian MARK/PAR1 family. We show
that KA1 domains from both yeast and human kinases bind
acidic phospholipids including PtdSer. For yeast Kcc4p, we
also present data using KA1 domain mutations that implicate
PtdSer as an important determinant for targeting this kinase to
its site of action at the bud neck.
Our findings with Kcc4p and Gin4p argue that—in addition to
its documented dependence on septin binding (Barral et al.,
1999; Longtine et al., 1998)—bud neck localization of septin-
associated kinases requires KA1 domain,phospholipid interac-
tions. On their own, neither the KA1 domain nor the septin-
binding region of Kcc4p/Gin4p/Hsl1p is sufficient for specific
bud neck targeting—but C-terminal fragments encompassing
both are efficiently localized to bud necks (Crutchley et al.,
2009; Longtine et al., 1998; Okuzaki and Nojima, 2001). Thus,
simultaneous engagement of the septin- and phospholipid-
binding domains appears to be required for Kcc4p, Gin4p, and
Hsl1p recruitment to septin assemblies at the bud neck for
kinase activation. This combination of septin-binding and phos-
pholipid-binding domains may function as an effective ‘‘coinci-
dence detector,’’ allowing the kinases to bind septins only at
membrane locations. The septins themselves also bind weakly
to anionic phospholipids (Casamayor and Snyder, 2003; Zhang
et al., 1999), suggesting further that kinase,phospholipid, kina-
se,septin, and septin,phospholipid interactions all cooperate
to organize a well-defined assembly at the bud neck. Coinci-
dence detection of this sort, in which multivalent interactions
involving both protein-binding and lipid-binding domains drive
complex formation, has been suggested for several systems
(Carlton and Cullen, 2005; Lemmon, 2008). It is particularly inter-
ing to a specific location (the bud neck) despite binding nonspe-
cifically to anionic phospholipids: it appears to restrict the ability
of Kcc4p to bind septins only in the context of a negatively
charged membrane surface, as a logical ‘‘AND’’ gate. Similar
coincidence detection mechanisms may also be relevant for
specific membrane targeting of human MARK/PAR1 family
proteins. Indeed, we show here that—like their structural coun-
terparts in the yeast septin-associated kinases—KA1 domains
of human MARK/PAR1 family proteins bind acidic phospholipids
in cells and in vitro.
Several reports have suggested that the C-terminal tail of
MARK/PAR1 kinases (which includes the KA1 domain) plays
a role in reversible autoinhibition of kinase activity (Elbert et al.,
2005; Marx et al., 2010; Timm et al., 2008). For example, the
C-terminal KA1 domain-containing region of the S. cerevisiae
Kin1 and Kin2 kinases was reported to interact with the
N-terminal catalytic domain (Elbert et al., 2005)—suggesting
direct intramolecular autoinhibitory interactions. A similar model
was also proposed for S. cerevisiae Hsl1p (Hanrahan and
Snyder, 2003), and septins were suggested to activate Hsl1p
by binding close to the C-terminal region and disrupting autoin-
hibitory intramolecular interactions. One concern raised about
this model (Crutchley et al., 2009; Szkotnicki et al., 2008) is
that it cannot explain why Hsl1p is activated only by assembled
septins at the bud neck, and not by free septin complexes.
Our findings provide an explanation: that the C-terminal region
of Hsl1p (and other septin-associated kinases) must bind to
both septins and anionic membrane phospholipids (via its KA1
domain) to drive the protein to the bud neck and relieve the
proposed intramolecular autoinhibition.
ing one or more phospholipid-binding domains is a recurring
themeinkinase regulation, with proteinkinase C(PKC)and other
AGC kinases providing well-characterized examples (Newton,
2009). Our studies suggest that the mechanistic role of the
KA1 domain in septin-associated kinases may be broadly anal-
ogous to that of C1 and C2 domains in PKC or the PH domain
in Akt (Newton, 2009). The KA1 domain lacks the lipid selectivity
of these other modules but appears to restrict specific recogni-
tion of other targets (such as septins) to a membrane context.
Extending our observations to the MARK/PAR1 family kinases,
the KA1 domain was previously implicated as a determinant of
membrane localization for MARK3 (Go ¨ransson et al., 2006),
and dissociation of hMARK2 from the plasma membrane coin-
cides with reduced activity (Hurov et al., 2004). Thus, phospho-
lipid engagement of the KA1 domain may also play a role in the
activation of these kinases at particular membrane locations.
Intriguingly, the KA1 domain fold has recently been seen in addi-
tional kinase contexts that warrant further investigation. A
C-terminal domain in the Arabidopsis AtSOS2 kinase has a KA1
domain fold (Sa ´nchez-Barrena et al., 2007) and includes a pro-
tein phosphatase-interacting (PPI) motif (in strand b1 and helix
a1). It is not known whether this domain binds phospholipids.
A C-terminal domain in the a subunit of heterotrimeric AMPK or-
thologs also has a KA1 domain fold and is intimately associated
with the C-terminal region of the bsubunit (Townley and Shapiro,
in a wide range of diseases, from Alzheimer’s disease to cancer
to diabetes, understanding the regulatory role of this domain is
an important goal. Our studies show that at least a subgroup
of KA1 domains bind nonspecifically to acidic phospholipids
and allow kinase activation to be coordinated with membrane
association, in an unexpected variation of a theme used by other
kinases that employ C1, C2, PH, and other domains.
Ras Rescue Assay
Ras rescue assays were performed exactly as described (Yu et al., 2004).
Briefly, DNA-encoding candidate proteins or fragments were PCR amplified
from S. cerevisiae (BY4741) genomic DNA or a HeLa cell cDNA library and
subcloned into modified p3S0BL2 (Isakoff et al., 1998) to generate plasmids
encoding Ha-Ras Q61Lfusions. Plasmidsweretransformedintocdc25tsyeast
For yeast studies, DNA fragments encoding candidate proteins or domains
were subcloned into modified pGO-GFP (Cowles et al., 1997) and transformed
974 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
2002). Images were collected at 1003 magnification using a Leica-DMIRBE
microscope and processed using Volocity deconvolution software (Improvi-
sion). All images of yeast cells are representative of >90% of cells expressing
the relevant GFP fusion protein (from over 100 cells in at least three experi-
ments). Analysis of full-length (or DKA1) Gin4p was performed in YEF1238
gin4D::TRP1 (YEF473A) cells (Longtine et al., 1998). To quantify plasma
membrane localization, lines were drawn across individual cells using ImageJ
andmean valuesfor fluorescence inthe plasma membrane (FPM)and cytosolic
(FCyt) regions were determined along the length of these lines as described
(Szentpetery et al., 2009). The ratio of these means (FPM/FCyt) was used as
a measure of plasma membrane localization.
For analysis of subcellular localization in mammalian cells, domains of
interest were subcloned into pEGFP-C1 (Clontech) and transiently transfected
into HeLa cells using Lipofectamine 2000 (Invitrogen). Cells were imaged at
403, and images processed as above. All microscopy images presented are
representative of at least three independent experiments, assessing over
100 cells each.
Surface Plasmon Resonance and Phospholipid Binding
Phospholipid-binding experiments were performed using surface plasmon
resonance (SPR) exactly as described previously (Yu et al., 2004) or sedimen-
tation assays (Kavran et al., 1998). For SPR studies, vesicles contained dio-
leoylphosphatidylcholine (DOPC) alone or the noted percent (mole/mole) of
test lipid in a DOPC background and were immobilized on L1 sensor chip
surfaces (BIAcore). Purified test proteins were flowed over these surfaces at
a series of concentrations, determined by absorbance at 280 nm using calcu-
lated extinction coefficients. SPR signals for each experiment were corrected
for background (DOPC) binding and plotted against protein concentration to
yield binding curves that were fit to simple hyperbolae. Experiments were per-
formed in 25 mM HEPES, pH 7.4, containing 150 mM NaCl. For sedimentation
assays, brominated PC was used as the background lipid and experiments
were performed exactly as described (Kavran et al., 1998).
Protein Preparation, Crystallization, and Data Collection
DNA encoding the KA1 domains from Kcc4p (residues 917–1037) and MARK1
(residues 683–795), plus an N-terminal hexahistidine tag, were subcloned into
pET21a (Novagen) for expression in E. coli BL21 (DE3) cells. For generating
selenomethionine (SeMet)-containing Kcc4p-KA1 protein, a third methionine
was introduced by substitution at L936, and protein was produced from
B834(DE3) methionine auxotrophs in MOPS-based minimal medium supple-
mented with SeMet. Proteins were purified from cell lysates in three steps,
using Ni-NTA resin (QIAGEN), cation exchange chromatography, and a Super-
dex 75 size exclusion column (GE Healthcare). Crystals were grown at 21?C
(at 300–400 mM) and reservoir solutions. MARK1-KA1 crystals were obtained
from 0.1 M Na acetate, pH 4.6, containing 0.04 M CaCl2, and 15%–25%
(w/v) PEG 3350. Kcc4p-KA1 crystals were obtained both from 0.1 M HEPES,
pH 7.4, containing 0.2 M (NH4)2SO4plus 20% (w/v) PEG3350 (for both native
and SeMet protein) and from 1.0 M K/Na tartrate, 0.1 M Tris, pH 7.0, with 0.2 M
LiSO4. Crystals were cryo-protected by direct transfer into reservoir solution
containing 15% (w/v) glycerol and were flash frozen in liquid nitrogen. Data
were collected at the Advanced Photon Source (Argonne, IL) beamlines
23ID-D and 23ID-B or the Cornell High Energy Synchrotron Source (CHESS)
beamline F2 and were processed using HKL2000 (Otwinowski and Minor,
Structure Determination and Refinement
Experimental phase information was obtained for Kcc4p-KA1 using data
collected from the SeMet-containing Kcc4p-KA1/L936M crystals, with
single-wavelength anomalous diffraction (SAD) methods implemented in
SHELX C/D/E (Schneider and Sheldrick, 2002). The resulting experimentally
phased map was excellent and allowed all but the first eight amino acids
(including the His6tag) to be traced with Coot (Emsley and Cowtan, 2004).
for datasets obtained with native protein using the program Phaser (CCP4,
1994). For MARK1-KA1, the structure was solved using MR with a search
model based on the mouse MARK3 KA1 domain NMR structure (PDB ID
1UL7) (Tochio et al., 2006), using Phaser (CCP4, 1994). Model building em-
ployed Coot (Emsley and Cowtan, 2004), following each round of refinement
using Refmac (CCP4, 1994) and PHENIX (Adams et al., 2010). Data collection
and refinement statistics are presented in Table S3. Structure figures were
generated using PyMol (DeLano, 2002).
Coordinates and structure factors have been deposited in the Protein Data
Bank (http://www.rcsb.org/pdb) with identification numbers 3OSE (MARK1-
KA1), 3OSM (Kcc4p-KA1 with bound tartrate), and 3OST (Kcc4p-KA1 with
figures, and three tables and can be found with this article online at doi:10.
We thank members of the Lemmon, Ferguson, and Bi laboratories and Ben
Black, Jim Shorter, and Greg Van Duyne for constructive comments. Erfei
Bi, Scott Emr, and Daryll DeWald provided yeast strains used in this study.
Crystallographic data were collected in part at the GM/CA Collaborative
Access Team at the Advanced Photon Source (APS), funded by NCI (Y1-
CO-1020) and NIGMS (Y1-GM-1104). Use of APS was supported by the
U.S. Department of Energy, under contract No. DE-AC02-06CH11357. Addi-
tional crystallographic data were collected at beamline F2 at the Cornell
High Energy Synchrotron Source (CHESS), supported by NIGMS and the
NSF (under award DMR-0936384), using the Macromolecular Diffraction at
CHESS (MacCHESS) facility, supported by the NIH (award RR-01646). This
toral fellowship from the American Heart Association Great Rivers Affiliate
Received: March 19, 2010
Revised: August 3, 2010
Accepted: November 1, 2010
Published: December 9, 2010
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Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. 977
EXTENDED EXPERIMENTAL PROCEDURES
Lipid Overlay Assay
GST fusion proteins of candidate proteins or their fragments were generated using a modified pGSTag vector as described (Klein
et al., 1998). GST fusions were labeled with32P and used in lipid overlay assays exactly as described (Kavran et al., 1998) to assess
binding to nitrocellulose-bound phosphoinositides and other anionic phospholipids.
Analysis of GFP/KA1 Domain Localization in Mutant Yeast Strains
To assess effects of altered phosphoinositide or PtdSer metabolism, the following mutant yeast strains were employed: mss4ts-
AA107, stt4ts-AA102, pik1ts-AA104, stt4ts/pik1ts-AA105 (Audhya et al., 2000) and cho1D BY4743 (Open Biosystems). For tempera-
ture-sensitive yeast mutants, cells were prepared at the permissive and restrictive temperatures as described (Audhya and Emr,
Audhya, A., and Emr, S.D. (2002). Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade. Dev. Cell 2, 593–
Audhya, A., Foti, M., and Emr, S.D. (2000). Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle
membrane dynamics. Mol. Biol. Cell 11, 2673–2689.
Cherry, J.M., Ball, C., Weng, S., Juvik, G., Schmidt, R., Adler, C., Dunn, B., Dwight, S., Riles, L., Mortimer, R.K., and Botstein, D. (1997). Genetic and physical
maps of Saccharomyces cerevisiae. Nature 387 (6632, Suppl) 67–73.
Cho, W., and Stahelin, R.V. (2005). Membrane-protein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struct. 34, 119–151.
Cunningham, C.C., Vegners, R., Bucki, R., Funaki, M., Korde, N., Hartwig, J.H., Stossel, T.P., and Janmey, P.A. (2001). Cell permeant polyphosphoinositide-
binding peptides that block cell motility and actin assembly. J. Biol. Chem. 276, 43390–43399.
Demel, R.A. (1994). Monomolecular layers in the study of biomembranes. Subcell. Biochem. 23, 83–120.
Desrivie `res, S., Cooke, F.T., Parker, P.J., and Hall, M.N. (1998). MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cyto-
skeleton in Saccharomyces cerevisiae. J. Biol. Chem. 273, 15787–15793.
Gouet, P., Courcelle, E., Stuart, D.I., and Me ´toz, F. (1999). ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308.
membrane is essential for yeast cell morphogenesis. J. Biol. Chem. 273, 15779–15786.
domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J. 17, 5374–5387.
Kavran, J.M., Klein, D.E., Lee, A., Falasca, M., Isakoff, S.J., Skolnik, E.Y., and Lemmon, M.A. (1998). Specificity and promiscuity in phosphoinositide binding by
pleckstrin homology domains. J. Biol. Chem. 273, 30497–30508.
Klein, D.E., Lee, A., Frank, D.W., Marks, M.S., and Lemmon, M.A. (1998). The pleckstrin homology domains of dynamin isoforms require oligomerization for high
affinity phosphoinositide binding. J. Biol. Chem. 273, 27725–27733.
Marsh, D. (1996). Lateral pressure in membranes. Biochim. Biophys. Acta 1286, 183–223.
Medina,O.P.,So ¨derlund,T.,Laakkonen,L.J.,Tuominen,E.K.,Koivunen, E.,andKinnunen,P.K.(2001).Binding ofnovelpeptideinhibitors oftypeIVcollagenases
to phospholipid membranes and use in liposome targeting to tumor cells in vitro. Cancer Res. 61, 3978–3985.
Stahelin, R.V., Karathanassis, D., Murray, D., Williams, R.L., and Cho, W. (2007). Structural and membrane binding analysis of the Phox homology domain of
Bem1p: basis of phosphatidylinositol 4-phosphate specificity. J. Biol. Chem. 282, 25737–25747.
Stefan, C.J., Audhya, A., and Emr, S.D. (2002). The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate.
Mol. Biol. Cell 13, 542–557.
Tochio, N., Koshiba, S., Kobayashi, N., Inoue, M., Yabuki, T., Aoki, M., Seki, E., Matsuda, T., Tomo, Y., Motoda, Y., et al. (2006). Solution structure of the kinase-
associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3. Protein Sci. 15, 2534–2543.
Yu, J.W., Mendrola, J.M., Audhya, A., Singh, S., Keleti, D., DeWald, D.B., Murray, D., Emr, S.D., and Lemmon, M.A. (2004). Genome-wide analysis of membrane
targeting by S. cerevisiae pleckstrin homology domains. Mol. Cell 13, 677–688.
Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., et al. (2001). Global analysis of protein
activities using proteome chips. Science 293, 2101–2105.
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. S1
Membrane Targeting Domains
No Phospholipid Binding
Figure S1. Identification of Phospholipid-Binding Domains in S. cerevisiae Proteins, Related to Figure 1
(A) Ras Rescue data for the 33 S. cerevisiae proteins (of 62 tested) capable of recruiting Ha-RasQ61L to yeast cell membranes (see also Table S1A). Serial dilu-
tions of cdc25tsyeast cultures expressing the noted protein fused to non-farnesylated Ha-RasQ61L were spotted onto duplicate selection plates lacking leucine
and incubated at the permissive (25?C: left) and restrictive (37?C: right) temperature for 4–5 days. Controls are shown at the top. The phospholipase C-d1PH
domain (PLCd-PH), binds PtdIns(4,5)P2with high affinity and serves as a positive control (Yu et al., 2004). The dynamin PH domain binds phospholipids very
weakly, and serves as a negative control (Isakoff et al., 1998; Yu et al., 2004).
All 33yeastproteinslisted inthisfigure gave positive Rasrescue results.Thosewithin the yellowbox allowed subsequent identificationofsubregionsresponsible
for phospholipid binding (see B). Those in the cyan box bound phospholipids in overlay experiments, and subdomains responsible could be identified in a few
cases (see B). Those listed in the green box failed to bind phospholipids in overlay or other experiments. Those in the gray box could not be expressed in suffi-
ciently large quantities for in vitro analysis of phospholipid binding.
(B) In addition to the Kcc4p KA1 domain (Figure 1), we identified subregions for Cam1p, Dps1p, Stp22p, and Rgd1p that appear necessary and sufficient for
membrane targeting. For Cam1p, this is a GST domain. For Stp22p, the N-terminal 190 amino acids, which constitute an UBC-like domain, appear sufficient
for membrane targeting. For Rgd1p, the amino-terminal 324 amino acids (an F-BAR domain) are necessary and sufficient for membrane targeting.
S2 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
Figure S2. Structure-Based Sequence Alignment of Human and Yeast KA1 Domains, Related to Figure 2
Mammalian and yeast KA1 domains are aligned, with sequence numbers provided and secondary structure elements listed above the sequence. The structure-
based sequence alignment was generated using ESPript (Gouet et al., 1999). The alignment of MARK/PAR1/Kin kinase KA1 domains closely resembles that re-
ported following determination ofthemMARK3-KA1 structure (Tochio etal.,2006).Alignment oftheKcc4p, Gin4p,and Hsl1pKA1domains is basedonstructural
studies described here. Red residues within vertical boxes represent positions at which amino acid type is most well conserved across the alignment.Secondary
structure elements aN (seen only in Kcc4p-KA1), a1, a2 and b1-b5 are marked. TT denotes a b turn. Sequence similarity is greatest toward the C terminus, espe-
cially in the b5/a2 region, which constitutes a major binding site for anions (Figure S4) at which we argue phospholipids are likely to bind. Residues in Kcc4p-KA1
thatcontribute tothebindingsiteforSO4#1,tartrate,orglycerol,and contributetomembraneassociation (see Figures 6andFigureS4)areboxedingreen.Those
thatcontributetothebinding sitefor SO4#2(notconservedinhMARK1-KA1)are boxedinorange. Residues inhMARK1-KA1thatformpart ofthebasic patch and
contributetomembraneassociation (Figure6)arealso coloredgreen.TheselieinareassimilartotheKcc4p-KA1 SO4#1bindingsite.Notetheconserved positive
charge in b5 and the relatively conserved S/T residues in a5 that contact glycerol in Kcc4p-KA1.
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. S3
Figure S3. Membrane Targeting of Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518in Yeast Strains with Altered Phosphoinositide Levels,
Related to Figure 3
Localization of GFP-fused Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518was examined in yeast strains with temperature sensitive mutations in PtdIns 4-
kinases (stt4tsand pik1ts) or the major PtdIns4P 5-kinase (mss4ts). Cells were grown to mid-log phase and then incubated for 40 min at either 26?C or 37?C.
The Num1p and Osh1p PH domains were used as control probes for PtdIns(4,5)P2and Golgi PtdIns4P respectively (Yu et al., 2004).All images are representative
of at least 75% of expressing cells observed.
PtdIns(4,5)P2levels are reduced in mss4tscells by approximately 40% at 26?C, and by more than 80% above the restrictive temperature (Stefan et al., 2002),
because of a mutation in the gene encoding the major PtdIns4P 5-kinase (Desrivieres et al., 1998; Homma et al., 1998). Despite these reductions in PtdIns
(4,5)P2levels, plasma membrane localization of the Kcc4p, Gin4p or Hsl1p C-termini is retained with no significant alteration in FPM/FCytratios above the restric-
tive temperature, whereas the PtdIns(4,5)P2-binding Num1p PH domain becomes delocalized (Yu et al., 2004). Similarly, temperature-sensitive mutations in the
genes encoding the PtdIns 4-kinases that generate PtdIns4P at the plasma membrane (Stt4p) and Golgi (Pik1p) respectively (Audhya et al., 2000) did not result in
altered FPM/FCytratios for the Kcc4p, Gin4p or Hsl1p C-termini above the restrictive temperature, whereas control phosphoinositide-dependent membrane
probes (Num1p and Osh1p PH domains) became delocalized. Thus, phosphoinositides do not appear to play a dominant role in membrane localization of
the C-terminal domains from yeast septin-associated kinases, despite the fact that our focus on Kcc4p came from its initial identification as a phosphoinosi-
tide-binding protein (Zhu et al., 2001).
S4 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
Figure S4. Details of a Potential Phospholipid-Binding Site in the Kcc4p KA1 Domain, Related to Figure 4
The structure of the Kcc4p KA1 domain was obtained from two different crystal forms, grown with sulfate and tartrate as the most prevalent anions (in space
groups P21and P1 respectively: see Table S3). Bound anions were seen in both structures, and were located in a common binding site between b5 and a1
that is therefore implicated as a phospholipid-binding site (see Figure 6).
(A) The Kcc4p-KA1 structure shown in Figure 4 and Figure 6 has two bound sulfate ions (SO4#1 and SO4#2) and glycerol molecules in the model. One sulfate
(SO4#1) lies close to the linker between strand b5 and helix a2, and is adjacent to a bound glycerol. This region of the KA1 domain is among the most conserved,
and was previously suggested on that basis to represent a binding site in hMARK3-KA1 (Tochio et al., 2006). Side chains of basic residues (K932, K1010, and
K1016) interact with the bound sulfate, and the S1014 and T1015 side chains interact with the bound glycerol.
(B)When crystallized inthepresenceof tartrate, atartrate ion isseen atthesame location, makingthe sameinteractionsas theglycerolmolecule (withS1014 and
T1015), plus someof those made by SO4#1 in (A). In addition, the tartrate ion interacts with R988 from the b3/b4 loop. The binding mode of tartrate(or sulfate plus
glycerol) suggests that this site represents a phospholipid-binding site, as also indicated by mutational studies presented in Figure 6.
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. S5
Figure S5. Localization of Human MARK/MELK KA1 Domains in Yeast Cells with Altered Phosphoinositide Levels, Related to Figure 5
Localization of the human MARK1, MARK3, and MELK KA1 domains (fused to GFP) was analyzed inmss4tscells, stt4tscells, and stt4ts/pik1tscells, grown to mid-
log phase, and then incubated for 40 min at either 26?C or 37?C before being examined by fluorescence microscopy. Images are representative of >90% of cells
observed(from three experimentsineachof which> 100cells wereanalyzed). Asdescribed inFigure S3,levels ofPtdIns(4,5)P2aregreatly reducedinmss4tsand
stt4tscells at the restrictive temperature. Plasma membrane and total PtdIns4P levels are greatly reduced at the restrictive temperature in stt4tsand stt4ts/pik1ts
cells respectively. In no case did elevating the temperature above the restrictive temperature lead to a significant alteration of FPM/FCytratios for the GFP/KA1, by
contrast with the impaired plasma membrane localization seen in cho1D cells in Figure 5.
S6 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.
Surface pressure change (mN/m)
Initial surface pressure (mN/m)
Figure S6. Monolayer Penetration of Kcc4p-KA1 and In Vitro Phospholipid Binding of hMARK1-KA1, Related to Figure 6
(A) Monolayer studies were performed using a mTroughS Langmuir trough (Kibron Inc., Helsinki, Finland) essentially as described (Cunningham et al., 2001;
Medina et al., 2001). Briefly, 1 ml of buffer (25 mM HEPES, pH 7.5, containing 150 mM NaCl) was placed as subphase solution in a well, and the desired lipid
mixtures were spread at the air-buffer interface in chloroform to different initial surface pressures. The resulting monolayers were allowed to equilibrate for 30
min before addition of the KA1 domain (His6Kcc4p901-1037) to a final concentration of 0.5 mM into the subphase solution. The increase in surface pressure
was then monitored for 30 min with constant stirring of the subphase following protein addition, with maximal change in surface pressure typically being reached
after 15 min. Lipid mixtures used were: pure stearoyl-oleoyl phosphatidylcholine (SOPC, gray points), SOPC containing 20% (mole/mole) SOPS (red points) or
SOPC containing 10% (mole/mole) PtdIns(4,5)P2(blue points). The increase in surface pressure achieved by addition of Kcc4p-KA1 (vertical axis) is plotted for
aseriesofinitial surfacepressures (horizontal axis). ThecriticalsurfacepressureQ
1996). Thus, Kcc4p-KA1 insertion into pure PC membranes is unlikely in physiological membranes. However, critical surface pressure values of 30.7 ± 0.3 mN/m
and 30.4 ± 0.4 mN/m were measured for SOPS/SOPC and PtdIns(4,5)P2/SOPC monolayers respectively, suggesting that the Kcc4p KA1 domain will insert into
membranes with these compositions in vivo. TheseQ
(B) Analysis of in vitro phospholipid-binding by hMARK1-KA1 variants tested in Figure 6B. SPR binding curves were performed for binding to negatively charged
membranes containing 10% (mole/mole) PtdIns(4,5)P2in a DOPC background. Curves shown are representative of at least two independent experiments. The
three mutants that showed impaired PtdIns(4,5)P2binding also failed to localize to the plasma membrane of yeast cells (Figure 6).
cisthesurfacepressure abovewhichmonolayerpenetration isnolongerseen.
For pure SOPCmembranes, this value was 25.5 ± 0.2 mN/m, whichis below the estimated cell membrane surface pressure of 30-35 mN/m (Demel, 1994; Marsh,
cvalues are similar to those previously reported for FYVE, PX, PH, ENTH, and other domains (Cho and
Stahelin, 2005; Stahelin et al., 2007).
Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc. S7
A Download full-text
Figure S7. Localization of GFP-Fused Kcc4p Harboring KA1 Domain Mutations, Related to Figure 7
(A) Wild-type or KA1 domain-mutated forms of intact Kcc4p fused to GFP were expressed in wild-type cells or cho1D cells (which lack PtdSer). A gallery of 6
representative budding cells is shown for each case, with DIC images at the left and epifluorescence images at the right of each pair. Neither the K1007S/
K1010S nor K1016S/K1020S double mutations in the KA1 domain were sufficient to prevent bud neck localization in wild-type cells, whereas the quadruple
K1007S/K1010S/K1016S/K1020S mutation abolished GFP/Kcc4p localization to the bud neck. Wild-type GFP/Kcc4p also retains bud neck localization in
cho1D cells, whereas the reduced plasma membrane charge in these cells was sufficient to cause delocalization of the GFP/Kcc4p variant harboring
a K1007S/K1010S double mutation. The behavior seen here represents that of more than 90% of cells imaged in each of three independent experiments
(>100 cells each).
(B) Western blotting analysis of yeast cell lysates using a GFP antibody (Covance) illustrates similar expression levels (with no significant free GFP) for wild-type
and mutated variants of GFP/Kcc4p. Samples were prepared by boiling ?10–20 ODs of cells grown to log phase in SDS-PAGE gel-loading buffer for 5–7 min
followed by SDS-PAGE and immunoblotting.
S8 Cell 143, 966–977, December 10, 2010 ª2010 Elsevier Inc.