Download Membrane nanodomains in plants: capturing form, function, and

Journal of Experimental Botany, Vol. 66, No. 6 pp. 1573–1586, 2015
doi:10.1093/jxb/erv054 Advance Access publication 27 February 2015
Review Paper
Membrane nanodomains in plants: capturing form, function,
and movement
Wiebke Tapken and Angus S. Murphy*
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA
* To whom correspondence should be addressed. E-mail: [email protected]
Received 24 November 2014; Revised 20 January 2015; Accepted 27 January 2015
Abstract
The plasma membrane is the interface between the cell and the external environment. Plasma membrane lipids
provide scaffolds for proteins and protein complexes that are involved in cell to cell communication, signal transduction, immune responses, and transport of small molecules. In animals, fungi, and plants, a substantial subset of
these plasma membrane proteins function within ordered sterol- and sphingolipid-rich nanodomains. High-resolution
microscopy, lipid dyes, pharmacological inhibitors of lipid biosynthesis, and lipid biosynthetic mutants have been
employed to examine the relationship between the lipid environment and protein activity in plants. They have also
been used to identify proteins associated with nanodomains and the pathways by which nanodomain-associated proteins are trafficked to their plasma membrane destinations. These studies suggest that plant membrane nanodomains
function in a context-specific manner, analogous to similar structures in animals and fungi. In addition to the highly
conserved flotillin and remorin markers, some members of the B and G subclasses of ATP binding cassette transporters have emerged as functional markers for plant nanodomains. Further, the glycophosphatidylinositol-anchored
fasciclin-like arabinogalactan proteins, that are often associated with detergent-resistant membranes, appear also to
have a functional role in membrane nanodomains.
Key words: Detergent-resistant membranes, membrane nanodomains, nanodomain function, ordered membrane domains,
plasma membrane ABC proteins, sphingolipids, sterols.
Introduction
Lipid membranes define the boundary of a cell, its organelles, and secretory subcompartments. They spatially separate incompatible biochemical processes, provide structural
support for transporters and receptors, function as exchange
surfaces for mineral ions and signalling molecules, and are
the initial point of contact for host–pathogen interactions.
Glycerolipids [mainly phospholipids (PLs)], sphingolipids (SLs), and sterols are the three main lipid classes that
constitute the plant plasma membrane (PM), and define
membrane organization and function. Recent combinatorial studies using high-resolution microscopy and lipid biosynthesis inhibitors suggest that proteins form specialized
laterally defined PM microenvironments characterized by
SLs and sterols (Lasserre et al., 2008, Raffaele et al., 2009;
Demir et al., 2014; Jarsch et al., 2014). These nanodomain
(ND) structures have been shown to increase the stability and
activity of embedded proteins and protein complexes: NDs
have been associated with receptor complex function (Sun
et al., 2002), transporter efficiency (Blakeslee et al., 2007;
Titapiwatanakun et al., 2009), channel regulation (Demir
et al., 2013), protein trafficking (Men et al., 2008; Yang et al.,
2013), and plant–bacterial interactions (Bhat et al., 2005;
Haney and Long, 2010; Lefebvre et al., 2010).
The terms lipid rafts and detergent-resistant membranes
(DRMs) are often used synonymously with NDs, largely for
historical reasons. The concept of lipid rafts was introduced to
describe membrane ‘patches’ in which discrete groups of proteins were localized in mammalian cells (Simons and Ikonen,
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1574 | Tapken and Murphy
1997). DRMs are physically isolated fractions of denser
sterol- and SL-enriched membranes, which are obtained
through extraction at 4 °C with non-ionic detergents, such
as Triton X-100 (Brown and Rose, 1992; Mongrand et al.,
2004; Borner et al., 2005; Martin et al., 2005). The term ‘lipid
raft’ emerged as a conceptualization of immiscible, steroland SL-rich ordered membrane domains with compositions
similar to those of DRMs. Co-purification of certain proteins with DRMs suggested that SLs and sterols could form
distinct sites of viral entry in mammalian cells (Nguyen and
Hildreth, 2000; Samuel et al., 2001; Bavari et al., 2002; Chazal
and Gerlier, 2003). This concept was later also applied to
plants, and thus DRM analyses paved the way for studies that
associated ordered membrane domains, or NDs, with physiological functions (Lasserre et al., 2008; Demir et al., 2013;
Yang et al., 2013).
Biochemical association of lipids and proteins with DRMs
does not directly establish the presence of ordered protein
and membrane complexes in plants any more than a sucrose
density gradient fractionation of membranes conclusively
demonstrates association of a particular protein with a specific cellular membrane structure. Interchangeable use of
the terms ‘lipid raft’ and DRM is, therefore, inappropriate.
Some authors suggest that the term ‘lipid raft’ should not be
used to describe any type of plant-associated ordered membrane domain and question that these domains occur in
plants (Tanner et al., 2011). This review does not in any way
imply that this metaphorical term should be used in the plant
literature.
A number of artefacts are generated in DRM preparation.
For instance, the strength of the non-ionic detergent used to
isolate DRMs can alter the composition of enriched sterols,
SLs, and associated proteins, and can also increase the membrane affinity of cytosolic proteins (Titapiwatanakun et al.,
2008; Tanner et al., 2011). More importantly, solubilization with Triton X-100 results in the temperature-dependent
restructuring of lipid domains, as observed in giant unilamellar vesicles (Heerklotz, 2002; Casadei et al., 2014). Thus,
DRM extraction is a selective isolation method and it is possible that the composition of functional NDs differs in living
cells. Due to its dependence on the isolation method used, the
lipid and protein composition of isolated DRMs is not identical to that of NDs. However, there is increasing evidence
that ordered membrane structures do exist in plants and that
they are functionally relevant.
Technological advances within the field of microscopy,
including new probes for atomic force microscopy (AFM),
super resolution confocal microscopy, high-resolution total
internal reflection fluorescence (TIRF), the variable angle
epifluorescence microscopy (VAEM) variant of TIRF used
in plants (Konopka and Bednarek, 2008), and enhanced
fluorescence recovery after photobleaching (FRAP), have
enabled more precise definition of the size and composition
of ND structures (Gutierrez et al., 2010; Truong-Quang and
Lenne, 2014). The identification of SL and sterol biosynthesis
mutants in Arabidopsis, in conjunction with these high-resolution imaging techniques, has provided a context for analyses of the contribution of these lipids to protein trafficking,
membrane structure, and physiology (Willemsen et al., 2003;
Men et al., 2008; Roudier et al., 2010; Markham et al., 2011;
Yang et al., 2013). As a consequence, ND size estimations
in plants have progressively decreased, from the micrometre
scale to the order of 10–100 nm, which is comparable with
large protein complexes, such as the cellulose synthase complex (Kimura et al., 1999; Cacas et al., 2012; Truang-Quong
and Lenne, 2013).
The wide range of ND sizes has spawned the use of the
‘microdomain’ and ‘nanodomain’ nomenclature to better
distinguish between differently sized complexes (Mongrand
et al., 2010; Demir et al., 2013). However, the terms still lack
concrete size delimitation. In summary, the current consensus is that the term DRM is utilized for lipids and proteins
that have been biochemically associated with SL- and sterolenriched membranes, and NDs as the functional assemblies
of lipids and proteins, when determined by biophysical or
microscopic techniques such as super resolution, TIRF,
or AFM.
In this review we will discuss the current understanding of
what determines protein association with NDs, where these
functional domains are formed, and how the lipid environment affects the trafficking of PM proteins. In particular, we
focus on the role of NDs in regulating the trafficking, localization, stability, mechanistic function, and interactions of PM
proteins, with an emphasis on ATP binding cassette (ABC)
transporters from the B and G subgroups in Arabidopsis.
Characteristics of the plant lipidome
Cholesterol and ergosterol are the most abundant sterols in
vertebrates and fungi, respectively (Shieh et al., 1977; Dupont
et al., 2012). Yeast DRMs are enriched in ergosterol (Bagnat
et al., 2000; Mongrand et al., 2004; Roche et al., 2008; Yang
and Murphy, 2009), while plant DRMs are mostly enriched in
the primary PM sterols β-sitosterol and campesterol. The sterols of the Arabidopsis PM and DRM fractions contain 80%
β-sitosterol and 10% campesterol, with no measureable stigmasterol content (Kierszniowska et al., 2009). Stigmasterol
is more highly abundant in the PM and DRMs from oat
and maize roots (Grandmougin et al., 1989; Norberg and
Liljenberg, 1991; Borner et al., 2005). Sterol concentrations in
PM and DRM fractions from tobacco bright yellow 2 (BY2)
cell cultures contain ~50% stigmasterol, ~20% 24-methyl
cholesterol, ~14% cholesterol, and only ~14% sitosterol
(Mongrand et al., 2004). Plant sterols can also be conjugated with sugars, which in turn can be fatty acylated to form
steryl glycosides and acyl-steryl glycosides. Steryl glycosides
and acyl-steryl glycosides are enriched in Arabidopsis DRMs
(Laloi et al., 2007; Minami et al., 2008). Experiments with
artificial membranes suggest that the lipid diversity in plants
decreases the temperature sensitivity and enhances lipid affinity for lipid raft formation (Xu et al., 2001; Beck et al., 2007).
Sphingosine and its derivative sphingomyelin are the most
common backbones of animal SLs (Cacas et al., 2012), but
are largely absent in plants. Instead, the hydroxysphinganine phytosphingosine is the primary plant SL backbone. In
Plant nanodomains and protein function | 1575
place of the glycosylceramides that are abundant in mammals, glycosyl inositol phosphoceramides (GIPCs) are the
predominant SLs in plants (Sperling et al., 2005; Pata et al.,
2010; Blaas and Humpf, 2013). GIPC formation requires the
enzyme inositol phosphorylceramide glucuronyltransferase1
(IPUT1), which is localized to the Golgi (Rennie et al., 2014).
GIPCs are highly abundant in membranes of Arabidopsis
leaves (Markham et al., 2006) and are also enriched in DRMs
(Borner et al., 2005). However, tobacco BY2 cell cultures are
more enriched in glycosphingolipids (Mongrand et al., 2004).
Hydrogen bonding between membrane-intercalated sterols
and either long chain bases (fatty acids) or large hydrophobic
head groups of SLs are energetically favoured over interactions with PL (Lingwood and Simons, 2010). This increases
the potential for SL/sterol partitioning and lateral immiscibility, which forces them into a denser, liquid-ordered state. The
long SL fatty acid chains confer a higher melting temperature (Brown and London, 1998) and are thought to increase
the thickness of the PM in the presence of sterols. However,
although largely accepted, the latter might in fact result from
the presence of proteins in these lipid environments, rather
than the presence of sterols (Mitra et al., 2003). Chemical
stripping experiments have shown that NDs are dependent
on the presence of sterols and SLs, and therefore the rigidity
of the liquid-ordered state is assumed to be the basis of the
structured nature of ND-associated proteins and their potential to associate into highly stable protein complexes.
Proteins localized to NDs
Although DRMs and NDs should not be thought of interchangeably in the context of their biochemical and biophysical properties, proteomic analyses of DRMs have provided
important information about proteins that could potentially
associate with NDs through their affinity for sterols and
SLs (Borner et al., 2005; Morel et al., 2006; Minami et al.,
2008; Fujiwara et al., 2009; Takahashi et al., 2012, 2013).
These proteomic exercises have been the jumping-off point
for studies seeking to relate NDs to specific functions such
as signal transduction (Demir et al., 2013; Srivastava et al.,
2013; Matsui et al., 2014; Zauber et al., 2014), substrate
transport (Sutter et al., 2006; Blakeslee et al., 2007; Yang
et al., 2013), stress responses (Beck et al., 2007; Minami et al.,
2008; Li et al., 2011), and pathogen responses (Bhat et al.,
2005; Raffaele et al., 2009; van der Meer-Janssen et al., 2010;
Bozkurt et al., 2014). A non-exhaustive list of ND and/or
DRM-localized proteins referenced herein can be found in
Table 1.
For example, the inward-rectifying potassium channel
KAT1 is a guard cell-localized protein, which has been implicated in abscisic acid- (ABA) dependent stomatal movement
(Sutter et al., 2007). KAT1 function at the guard cell PM is
dependent on syntaxin of plants 121 (SYP121/PEN1), and
KAT1 forms large protein aggregates of ~500 nm in diameter, consisting of an estimated 50 homo-tetramers (Sutter
et al., 2006; Reuff et al., 2010). Lateral movement of KAT1
at the PM is very slow, as observed through pulse–chase-like
strategies in conjunction with kymographic analyses (Sutter
et al., 2006). Another stomatal protein, slow anion channel homolog 3 (SLAH3), interacts with calcium dependent
protein kinase 22 (CPK21) in guard cell NDs (Demir et al.,
2013). ABA binding to the receptor regulatory components
of ABA receptor1/PYR1 (pyrobactin resistance 1)-like proteinc9 (RCAR/PYL9) results in recruitment of the ABA
insensitive 1 (ABI1) protein phosphatase 2C to the SLAH3/
CPK21 complex and rapid activation of the anion channel.
It appears that the ordered ND environment is in this case
essential to complex formation and function.
SYP121, also known as PENETRATION1 (PEN1),
appears to function in rapid recruitment of proteins to NDs.
SYP121 is recruited to sites of Blumeria graminis (powdery
mildew) infection in Arabidopsis, where it is required for
papillae formation on the apoplastic side of the PM (Assaad
et al., 2004). In unchallenged cells, SYP121 is continuously
cycled between the endomembrane system and the PM, but
rapidly partitions into NDs after pathogen infection (Bhat
et al., 2005; Nielsen et al., 2012), in a manner similar to what
is observed with KAT1 in guard cells.
Plasma membrane intrinsic protein2;1 (PIP2;1/PIP2A) is a
member of the aquaporin family that facilitate water movement across the PM (Santoni et al., 2003; Katsuhara et al.,
2008). PIP2;1 partitions into DRMs (Demir et al., 2013) and
its association with NDs has been investigated in great detail
(Li et al., 2011). In control conditions, PIP2;1 is more or less
homogenously distributed at the PM. However, under salt
stress (NaCl) it increasingly co-localizes with Flotillin1 in specific foci at the PM, which is accompanied by the reduction of
its lateral mobility (Li et al., 2011). This restriction was suggested to lead ultimately to PIP2;1 internalization from the
PM. PIP2;1 appears to be able to move in and out of NDs.
As PIP2;1 functions as an oligomer, this flexibility has been
suggested to facilitate protein–protein interactions at the PM
(Li et al., 2011). Notably, the NDs with which PIP2;1 associates are distinct from ABCB19, as FEN (a sterol biosynthesis
inhibitor) treatment does not alter PIP2;1 localization at the
PM (whereas ABCB19 is affected), and salt treatment did not
alter ABCB19 localization (Yang et al., 2013).
Emergence of standard ND marker
proteins
One highly conserved ND protein family, which was initially
identified in other kingdoms, has become a useful ND marker
in plants. In animal cells, flotillins are proteins of unknown
function that are associated with caveolae, which are steroland SL-rich membrane invaginations that mediate a variety
of cell to cell communication processes (Bickel, 1997; Babuke
and Tikkanen, 2007; Takeshita et al., 2012). Arabidopsis
Flotillin1 forms highly dynamic punctate structures at the
PM (Li et al., 2012). Plant flotillins appear to have a role in
symbiotic interactions with nitrogen-fixing bacteria (Haney
and Long, 2010).
Another important marker for NDs are the remorins,
which were originally identified in the Solanaceae (Farmer
1576 | Tapken and Murphy
Table 1. Proteins associated with liquid-ordered domains
Protein
Function
ND identified
(microscopy)
SL/sterol dependence in
DRMs
References
ABCB1
Auxin transport
–
Demir et al. (2013)
ABCB4
Auxin transport
Yes
Extracted with 1% Brij and
Triton X-100
SL dependent
ABCB19
Auxin transport
Yes
ABCB21
Auxin transport
–
ABCG36/PEN3
Pathogen response
–
Not MβCD-responsive
ABCG37/PIS1
–
FB1 and FEN treatment induce
no changes in PM localization
FLA2
Coumarin and auxinic
compound transport
Cell adhesion
–
Sterol dependent (MβCD);
extracted with 1% Brij and
Triton X-100
FLA proteins
Cell adhesion
Sterol dependent (MβCD)
Kierszniowska et al. (2009)
Flotillin1 and flotillins
Pathogen response
Yes
Sterol dependent (MβCD)
KAT1
Potassium channel
Yes
Not sensitive to MβCD
PIN1
Auxin transport
Yes
PIN2
Auxin transport
Yes
FB1 and tsc10a (SL) show
more intracellular structures;
cpi1-1 (sterol)
cpi1-1, cvp1-3, and fackel-J79
(sterol); DRM preparation
Borner et al. (2005); Haney and
Long (2010); Li et al. (2012);
Jarsch et al. (2014)
Sutter et al. (2006); Reuff et al.
(2010); Jarsch et al. (2014)
Blakeslee et al. (2007); Men et al.
(2008); Yang et al. (2013)
PIP2;1
Aquaporin
Yes
Sterol dependent (MβCD); not
sensitive to FB1 (SL) or FEN
(SL); extracted with 1% Brij and
Triton X-100
Remorin1.3
Pathogen response
Yes
Sterol dependent (MβCD);
extracted with 1% Brij and
Triton X-100
Remorins
Pathogen response
Yes
SLAH3
SYP121/PEN1
Nitrate, ABA signalling
Pathogen response
Yes
Yes
et al., 1989; Reymond et al., 1996). Remorins are plant-specific, peripheral membrane proteins that aggregate in clusters of ~70 nm in diameter (Raffaele et al., 2009). One of the
most commonly used ND markers, Remorin1.3, has been
shown to function in immune responses upon infection with
the Potato virus X and Phytophtora infestans (Perraki et al.,
2012; Bozkurt et al., 2014), but remorins remain otherwise
functionally uncharacterized. Flotillin and remorin isoforms
associate with distinct ND types (Jarsch et al., 2014), and are
therefore increasingly used to analyse functional relationships between proteins and specific NDs.
Other identified ND markers are KAT1, SLAH3 (mentioned above), and multiple members of the ABC transporter
family of the subclasses B and G. ABC transporters form a
superfamily of membrane transporters that is ubiquitously
Mainly SL, but also sterol
dependent
Extracted with 1% Brij and
Triton X-100
Sterol dependent
In structures marked by filipin
(sterol); in DRMs of rice
Borner et al. (2005);
Titapiwatanakun et al. (2009);
Yang et al. (2013)
Titapiwatanakun et al. (2009);
Yang et al. (2013)
Demir et al. (2013)
Minami et al. (2008);
Kierszniowska et al. (2009)
Yang et al. (2013); Fourcroy et al.
(2014)
Kierszniowska et al. (2009); Demir
et al. (2013)
Men et al. (2008); Pan et al.
(2009); Titapiwatanakun et al.
(2009)
Li et al. (2011); Demir et al. (2013);
Yang et al. (2013)
Raffaele et al. (2009); Demir et al.
(2013)
Mongrand et al. (2004); Lefebvre
et al. (2010); Jarsch et al. (2014)
Demir et al. (2013)
Assaad et al. (2004); Bhat et al.
(2005); Fujiwara et al. (2009)
found across kingdoms. Arabidopsis ABCs cluster into eight
subclasses on the basis of sequence homology and domain
organization (Verrier et al., 2008). Plant ABCs of the subclass
B transport organic acid compounds such as indole-3-acetic
acid (the principle auxin IAA; ABCB1, ABCB4, ABCB19,
ABCB21; Noh et al., 2001; Geisler et al., 2005; Terasaka
et al., 2005; Kamimoto et al., 2012), and malate and citrate
(ABCB14; Kaneda et al., 2011). The full-length ABCGs
ABCG36/PEN3 and ABCG37/PIS1 transport a greater range
of substrates, including xenobiotics (ABCG37; Ito and Gray,
2006), compounds involved in pathogen responses (ABCG36;
Stein et al., 2006), cadmium (ABCG36; Kim et al., 2007), and
the artificial auxin 2,4-dichlorophenoxyacetic acid (2,4-D)
(ABCG37; Ito and Gray, 2006) as well as the IAA precursor indole-3-butyric acid (ABCG36&ABCG37; Strader and
Plant nanodomains and protein function | 1577
Bartel, 2009; Růžička et al., 2010), and coumarin compounds
(ABCG37; Fourcroy et al., 2014). Although all of these
transporters have been associated with NDs and/or DRMs,
ABCB4, ABCB19, and ABCG37 are most commonly identified in these structures (Minami et al., 2008; Kierszniowska
et al., 2009; Demir et al., 2013; Yang et al., 2013).
The glycosylphosphatidylinositol (GPI)-anchored fasciclin-like arabinogalactan (FLA) proteins and some jacalin lectin proteins are recurrent residents of DRMs (Borner et al.,
2005; Kierszniowska et al., 2009; Demir et al., 2013) that are
also used as ND markers. The potential of these proteins
to mediate interactions of NDs with cell walls makes them
prime targets for investigation.
Determinants for ND association of
proteins
Protein association with DRMs depends on electrostatic
(hydrogen bonds) and hydrophobic interactions with the
lipids (Murray et al., 1997; Cacas et al., 2012). These interactions appear to contribute to the formation and stability of
functional NDs and associated protein complexes. An example is the interaction of the two Arabidopsis auxin transporters ABCB19 and PINFORMED1 (PIN1; Fig. 2). ABCB19
is associated with a subset of DRMs characterized primarily
by SLs, but also sterols (Titapiwatanakun et al., 2009; Yang
et al., 2013). PIN1 is a highly polarized transporter and is
the primary mediator of polar auxin streams required for
organogenesis and shootward auxin transport (Gälweiler
et al., 1998; Benková et al., 2003; Reinhardt et al., 2005;
Marhavý et al., 2011). PIN1 polar localization in some cell
types is destabilized in mutants that are deficient in sterol biosynthesis (Willemsen et al., 2003). ABCB19 and PIN1 have
been shown to interact in DRMs (Titapiwatanakun et al.,
2009), and ABCB19 stabilizes PIN1 in discrete domains
when the two proteins co-occur. Loss of ABCB19 in cells
where it otherwise would co-localize with PIN1 results in
reduced abundance of PIN1 at the PM (Blakeslee et al., 2007;
Titapiwatanakun et al., 2009; Yang et al., 2013). PIN1 was
also observed to exhibit increased intracellular abundance in
cells within the normal ABCB19 expression domain in both
the abcb19 mutant and the wild type, treated with sterol/SL
biosynthesis inhibitors and/or stripping reagents (Yang et al.,
2013). Co-purification of FLA2 with ABCB19 suggests that
the GPI-anchored FLA2 could be part of the ABCB19/PIN1
ND (Fig. 2). Finally, PM localization of ABCB19 itself was
substantially reduced in SL- and sterol-deficient mutants.
The sterol and SL dependence of ABCB19 transport function and PIN1 interaction has been demonstrated in heterologous systems and in planta. Auxin transport activity of
ABCB19 in heterologous systems (Schizosaccharomyces
pombe) is increased by addition of sterols (Titapiwatanakun
et al., 2009) or when ergosterol-containing membrane domains
are present (Yang and Murphy, 2009). When ABCB19 and
PIN1 are co-expressed, a synergistic increase in auxin transport and substrate specificity is observed (Blakeslee et al.,
2007), and loss of either ABCB19 or PIN1 increases polar
transport of benzoic acid (Blakeslee et al., 2007), which is
normally not transported in a polar manner.
GPI and S-acylation modifications are hallmarks of
ND-associated proteins in animals (Garner et al., 2007). GPI
anchoring and some S-acylation modifications appear in some
cases to be solely sufficient for conferring DRM partitioning
in plant cells (Borner et al., 2005; Sorek et al., 2007). About
1% of the Arabidopsis proteome encodes GPI-anchored proteins (Arabidopsis Genome Initiative, 2000; Borner et al.,
2003). GPI anchoring results in protein targeting to the exoplasmic leaflet in animals and plants (Ikonen and Simons,
1998). Failure to generate GPI anchors in the abnormal pollen
tube guidance 1 (aptg1) mutant aptg1 results in embryo lethality (Dai et al., 2014), highlighting the importance of GPIanchored proteins for plant development and reproduction.
Many proteins involved in cell to cell communication during seed coat and pollen development are over-represented
in the GPI proteome (Borner et al., 2003; Tsukamoto et al.,
2010; Edstam and Edqvist, 2014). One of these is COBRALIKE 10 (COBL10), a polar-localized GPI-anchored pollen
tube protein that interacts with female chemotropic signals to
direct growth within the pistil, leading to successful fertilization (Li et al., 2013). In aptg1 heterozygotes, COBL10 fails to
localize to the PM (Dai et al., 2014).
Plasmodesmata (PDs) connect neighbouring plant cells
and are crucial for facilitating the intercellular exchange of
nutrients and hormones, and to provide passageways for
pathogens. PDs are enriched in sterols and SLs, and many PD
proteins are represented in the GPI proteome (FernandezCalvino et al., 2011). Among these are members of the PD
callose binding protein (PDCB) family that adjusts PD pore
size by regulating the amount of callus deposition. PDCBs
have been experimentally verified to be GPI anchored
(Simpson et al., 2009).
S-Acylation is the post-translational addition of a palmitate or, less frequently, a stearate moiety to a cysteine residue
of the target protein. Recent proteomic studies suggest that
S-acylation is the most frequent lipid modification of proteins, with a total of 581 S-acylated proteins (~2% of the
proteome) identified in Arabidopsis (Hemsley et al., 2013;
Hemsley, 2014). For instance, some members of the remorin
family are S-acylated (Hemsley et al., 2013; Konrad et al.,
2014). Two acylating enzymes, protein S-acyltransferase
10 (PAT10), located in the Golgi and tonoplast, and TIP
GROWTH DEFECTIVE 1 (TIP1), localized to the PM,
acylate proteins in Arabidopsis (Hemsley et al., 2005; Zhou
et al., 2013; Qi et al., 2013). S-Acylation is the only fully
reversible lipid modification, making it particularly important in lipid signalling (Smotrys and Linder, 2004). Notable
examples of S-acylated proteins in Arabidopsis are members
of the large family of leucine-rich repeats (LRR) receptorlike kinases, soluble N-ethylmaleimide-sensitive factor
attachment protein receptors (SNAREs) such as SYP121/
PEN1, and members of the Rho-like GTPases in plants
(ROPs; Sorek et al., 2007; Craddock et al., 2012). GTPbound ROPs have been localized to DRMs and they regulate cell division, morphogenesis, and cell polarity (Yalovsky
et al., 2008). S-Acylation within the G-domain in ROP6 (at
1578 | Tapken and Murphy
which GTP binding and hydrolysis takes place) is vital for
its association with DRMs and, thus, probaby its function in
vivo (Sorek et al., 2007, 2010). Inactive, GDP-bound ROPs
can exhibit other lipid modifications such as prenylation, but
locate to the cytosol.
N-Myristoylation is the addition of the 14-carbon
saturated fatty acid myristate (myristoyl-CoA) through
N-myristoyltransferases (NMT) to an N-terminal glycine
of a target protein (Qi et al., 2000; Sharma, 2004; Martinez
et al., 2008). The myristate moiety interacts with negatively
charged phosphatidylserine or phosphatidylinositol phosphates in the endoplasmic leaflet to enhance protein affinity
for the PM (Resh, 2013). N-Myristoylation alone is not sufficient for DRM association in animals and plants, but tandem N-myristoylation in conjunction with single S-acylation
appears to provide a viable signal for DRM partitioning in
animal cells (Zacharias et al., 2002). The virulence of some
bacteria and viruses depends on this lipid modification, as
it confers PM association of their effector proteins, such
as members of the HopZ family from Pseudomonas syringae and AC4 of the East African cassava mosaic Cameroon
virus (Fondong et al., 2007; Lewis et al., 2007). However,
N-myristoylation of ND-associated proteins has not been
unequivocally demonstrated in plants.
Prenylation, like N-myristoylation, enhances the affinity
of proteins for the PM. C-15 farnesyl or C-20 geranylgeranyl
isoprenoid moieties are added by protein farnesyl transferase
(PFT) and protein geranyl-geranyltransferase-1 (PGGT-1),
respectively, to target proteins. Both enzymes consist of one
α-and one β-subunit, with the β-subunit conferring substrate
specificity (Maurer-Stroh et al., 2003). PFT and PGGT-1
recognize the motif cysteine-a1-a2-X, where ‘a’ corresponds
to aliphatic amino acids and X can be cysteine, methionine,
serine, glutamine, alanine, or leucine (Sorek et al., 2011). The
amino acid at the ‘X’ position generally determines whether
a protein is farnesylated (serine, glutamine, methionine,
alanine) or geranylgeranylated (leucine) (Reid et al., 2004;
Resh, 2006). However, to date, prenylated proteins have been
shown to associate exclusively with phospholipid domains.
A summary of software currently available to predict co-and
post-translational modifications is given in Table 2.
Recent evidence suggests that some protein structures could
directly confer protein association with NDs. Experiments
utilizing a fusion of yellow fluorescent protein (YFP) to the
C-terminus of potato Remorin1.3 (RemCA) containing an
α-helical structure that forms a tight amphipathic hairpin
within the lipid bilayer showed that this structure alone was
sufficient to target the protein to DRMs (Perraki et al., 2012).
Homologous peptides were subsequently identified in publicly available databases (Raffaele et al., 2013), but have not
been confirmed. Multiple members of the remorin family in
Arabidopsis are predicted and/or verified to be S-acylated at
C-terminal cysteine residues, which is not sufficient for their
interaction with the PM, but rather appears to enhance their
interaction with this lipid environment (Hemsley et al., 2013;
Konrad et al., 2014). A putative S-acylation site was also
predicted for Flotillin1 (Borner et al., 2005), but still awaits
experimental confirmation.
Sterol and SL inhibitors and dyes used to
analyse ND function
Sterol and SL biosynthetic inhibitors have been extensively used to dissect ND function (Laloi et al., 2007;
Titapiwatanakun et al., 2009; Li et al., 2011; Markham et al.,
2011; Yang et al., 2013). The effects of pharmacological
inhibitors can be controlled in a spatiotemporal manner that
increases the chances to pinpoint the involvement of specific
lipid species in ND formation. In combination with microscopic techniques, dyes allow for the direct visualization of
the effects of sterol and SL depletion in real-time. However,
care must be taken to avoid indirect or solvent effects on membrane integrity that increase marker protein degradation.
Some of these inhibitors have been adopted from animal
and fungal studies (Baloch et al., 1984; Bagnat et al., 2000)
and can be used to distinguish the effects of structural sterol
depletion from those of the reduction of steroid hormones
Table 2. Searchable databases for protein modifications
Modification
Software
Source
Plant Specific
General
ScanProsite
ARAMEMNON
ExPASy
PredGPI
BIG_PI Plant Predictor
GPI-SOM
FragAnchor
CSS-Palm 2.0
PalmPred
NBA-Palm
NMT-The Myr Predictor
ExPASy Myristoylator
PlantsP
TermiNator3
PrePS
de Castro et al. (2006)
Schwacke et al. (2003)
Artimo et al. (2012)
Pierleoni et al. (2008)
Eisenhaber et al. (2003)
Fankhauser and Mäser (2005)
Poisson et al. (2007)
Ren et al. (2008)
Kumari et al. (2014)
Xue et al. (2006)
http://mendel.imp.ac.at/myristate/SUPLpredictor.htm
http://web.expasy.org/myristoylator/
Podell and Gribskov (2004)
Martinez et al. (2008)
Maurer-Stroh and Eisenhaber (2005)
No
Yes
No
Yes
Yes
Included
Included
No
No
No
No
Included
Yes
Included
No
GPI anchors
S-Acylation
N-Myristoylation
Prenylation
Plant nanodomains and protein function | 1579
(brassinosteroids) in plants (Yang et al., 2013). For example,
fenpropimorph (FEN) is a chemical inhibitor of sterol synthesis that targets the plant-specific, endoplasmic reticulum
(ER)-localized cycloeucalenol-obtusifoliol isomerase, but
also has some impact on glucosylceramide (SL) accumulation (Fig. 1; Rahier et al., 1989; Laloi et al., 2007; Men et al.,
2008). FEN treatment decreased the amount of Δ5-sterols,
glucosylceramide, and overall DRM content of PM fractions
from leek seedlings (Laloi et al., 2007). Propiconazol and
brassinolide are inhibitors of steroid hormone biosynthesis
that can be used to distinguish the effects of structural sterol
depletion from hormone signalling (Hartwig et al., 2012;
Yang et al., 2013).
Methyl-β-cyclodextrin (MβCD) can selectively bind
the small sterols in its hydrophobic cavity (Zidovetzki and
Levitan, 2007). Treatment of tissues with MβCD strips
membrane sterols, and, in most cases, releases or destabilizes
sterol-dependent membrane proteins (Ohvo and Slotte, 1996;
Zidovetzki and Levitan, 2007; Kierszniowska et al., 2009). It
is also possible that MβCD treatment induces endocytosis, as
is seen with Flotillin1 (Li et al., 2012). MβCD will displace
many ND proteins during transit through secretory compartments beginning at the smooth ER, the initial point of sterol
incorporation. However, short-term treatments can be used
to restrict MβCD stripping to the PM, and internalization of
PM proteins resulting from sterol depletion can be observed
within 30–60 min of the treatment (Li et al., 2012; Yang et al.,
2013). MβCD treatment has also been used as a tool in proteomic analyses to differentiate membrane proteins that partition into DRMs due to sterol association from other DRM
proteins (Kierszniowska et al., 2009). This study showed that
regulatory/signalling membrane proteins are less consistently found to be associated with DRMs compared with, for
instance, the FLAs.
Filipin is an antibiotic with sterol-binding fluorochrome
properties. Its fluorescent property (excitation 360 nm, emission 480 nm) makes it a useful sterol visualization agent
(Norman et al., 1972; Drabikowski et al., 1973). However,
filipin also disrupts sterol interactions and has been used
to interfere with internalization of PIN2 (Men et al., 2008).
Filipin also binds to PL when used at higher concentrations
(Drabikowski et al., 1973) and can introduce lesions in the
lipid bilayer, which could lead to unspecific internalization or
clustering of proteins (Santos et al., 1998).
The primary pharmacological SL biosynthesis inhibitor used in plant ND studies is the mycotoxin fumonisin B1
(FB1). FB1 inhibits ceramide synthase activity and has been
shown to interrupt SL biosynthesis in plants (Wang et al.,
1991; Abbas et al., 1994). However, as SLs are highly stable,
depletion of SLs from membranes after FB1 treatment is relatively slow (Yang et al., 2013). 1-Phenyl-2-palmitoylamino3-morpholino-1-propanol, which is a glycosylceramide
synthase inhibitor, is also used as an SL synthesis inhibitor
(Yang et al., 2013), but is thought to have multiple targets in
Fig. 1. Sites of alteration of lipid biosynthesis/content resulting from genetic lesions or pharmacological inhibition. Sphingolipid biosynthetic inhibitors
are marked in red, sterol inhibitors are blue, and trafficking inhibitors are in black. Short-term treatment with FEN and MβCD leads to the internalization
of sterol-dependent PM proteins. FEN-sensitive PM proteins have been found to be internalized to TGN/EE compartments (marked by ‘1st’) and
characterized by SYP61 (green spiked vesicle). After extended treatment with MβCD and FEN, internalized proteins can first be found in the PVC/MVB
(marked by ‘2nd’). The SYP61 compartment functions primarily in anterograde secretion of PM proteins (thicker arrow) but also receives a subset of
PM proteins after endocytosis. Nanodomain-associated ABCB proteins co-localize with so-called ‘endosin bodies’ marked by SYP61 that are formed
after treatment with ES-1 (Robert et al., 2008). ConcA, which induces fusion of PVC/MVB vesicles with TGN/EE vesicles, is marked by SYP61 and SLs.
The thiol protease inhibitor E-64d inhibits the fusion of SYP61 with the PVC/MVB in an SL-dependent manner. PVC/MVB, pre-vacuolar compartment/
multivesicular body; TGN/EE, trans-Golgi network/early endosome; ConcA, concanamycin A; ES-1, endosidin1; FEN, fenpropimorph; FB1, fumonisin B1.
Mutations: gcs, glycosylceramide synthase; pas1, pasticcino1; loh1,3: LAG1 homologue; tsc10a-2, temperature-sensitive CSG2 suppressor; cvp1-3,
cotyledon vascular pattern1; cpi1-1, cyclopropylsterol isomerase1, erh1, enhancing RPW8-mediated HR-like cell death, codes for IPCS.
1580 | Tapken and Murphy
plants. An overview of where the pharmacological inhibitors
and lipid stripping agents function in the plant cell is shown
in Fig. 1.
Use of sterol and SL biosynthetic mutants
to analyse ND function
An alternative to the use of sterol and SL biosynthetic inhibitors is the use of well-characterized sterol and SL biosynthesis mutants in Arabidopsis (Willemsen et al., 2003; Men
et al., 2008; Pan et al., 2009; Carland et al., 2010; Yang et al.,
2013). Biosynthetic mutants are particularly useful, as lesions
in genes encoding enzymes in the biosynthetic pathway can
eliminate a desired lipid species. For example, cotyledon vascular pattern 1 (cvp1) is defective in sterol methyl transferase
2 (SMT2) and contains low levels of structural sterols, but
not low levels of steroid hormones (Carland et al., 2010).
The cvp1-3 mutant develops in a relatively normal fashion
and sets seed. It also expresses lower levels of SMT1 and
SMT3, thus reducing structural sterol levels in the root epidermis where high- resolution light microscopy analyses of
fluorescent proteins and membrane dyes are most effective.
Studies of cvp1-3 have been used to identify defects in trafficking of sterol-associated proteins such as PIN2, ABCB4,
and ABCB19 (Pan et al., 2009; Yang et al., 2013). However,
the residual levels of structural sterols in cvp1-3 epidermal
cells and their contribution to PM protein stability are difficult to determine.
An alternative is the use of mutants with lesions in cyclopropylsterol isomerase1/cycloeucalenol-obtusifoliol isomerase (cpi1/coi) that is expressed in epidermal cells (Men et al.,
2008), and has been used to analyse trafficking of sterol-associated proteins (Men et al., 2008; Pan et al., 2009; Yang et al.,
2013). However, cpi1 mutants are severely stunted and are
defective in brassinosteroid biosynthesis (Men et al., 2008).
Further, levels of the minority structural sterols cycloeucalenol and 24-methylenecycloartanol are higher in cpi1 than in
the wild type, and potentially substitute for stigmasterol and
sitosterol, leading to false-negative results (Men et al., 2008;
Yang et al., 2013). Other mutants that have been utilized in
trafficking studies are fackel, a weak sterol C14 reductase
allele that is defective in later steps of sterol biosynthesis
and exhibits faulty stomatal precursor cell division (Jang
et al., 2000; Qian et al., 2013), and sterol methyltransferase
1 (smt1orc) that is defective in conversion of 24-methylenelophenol into citrostadienol, a precursor of stigmasterol, and
exhibits dwarfism and cotyledon defects (Willemsen et al.,
2003). Both of these mutants exhibit faulty PM localization
of PIN proteins (Willemsen et al., 2003; Pan et al., 2009).
Reduction of SL levels results in a visible effect on localization of ND-associated proteins. Reductions of very long
chain fatty acids that contribute to SL formation in pasticcino1 mutants and multiple SLs in lag1 homolog 1 and 3
result in reduced auxin transport and partial mislocalization of PIN1 (Roudier et al., 2010; Markham et al., 2011),
similar to what is observed in the abcb19 mutant. The temperature-sensitive CSG2 suppressor gene (tsc10a-2) encodes
a 3-ketodihydrosphinganine reductase, a SL biosynthesis
mutant that exhibits increased tricotyledon formation (Chao
et al., 2011; Yang et al., 2013) and phenotypes similar to
abcb19 (Yang et al., 2013). ABCB19 was shown to aggregate partially in compartments marked by SYP61 and the
V-SNARE VTI12 in tsc10a, and PIN1 exhibited mislocalization similar to that observed in abcb19 mutants.
Trafficking of ND-associated proteins
Plant sterols and SLs are initially synthesized in the smooth
ER and then are elaborated by glycosylation, acylation, and
lipidification during anterograde movement through the
secretory system (Bowles et al., 1977; Moreau et al., 1998;
Melkonian et al., 1999; Rennie et al., 2014). Analysis of membrane fractions from leek seedlings showed that DRMs are
only present in post-ER membrane compartments (Laloi
et al., 2007), and NDs appear first to assemble within the
Golgi and trans-Golgi network (TGN) (Laloi et al., 2007;
Klemm et al., 2009). However, the exact timing of formation
of specific types of NDs, the extent to which ND-associated
proteins help nucleate and maintain NDs, and the relative
roles of lipid species in the process remain unresolved.
These questions are increasingly being examined in live
imaging studies using variants of laser scanning confocal
and TIRF microscopy in combination with lipid dyes, pharmacological inhibitors, mutants, and subcellular trafficking
markers. In these studies, ND–protein associations have been
confirmed to occur primarily in post-Golgi compartments,
especially at the TGN/early endosome (EE), where exocytotic
and endocytotic events co-occur (Dettmer et al., 2006). For
example, PM abundance of the auxin transporter PIN2 is
regulated by both secretion and endocytosis from the PM.
In the sterol biosynthesis mutant cpi1-1, an increase of PIN2
at the PM is detected (Men et al., 2008). FRAP analysis in
the presence of the vesicle trafficking inhibitor brefeldin
A (BFA) suggested that the increase of PIN2 at the PM is
due to reduced endocytosis of the protein. This regulation is
consistent with the observed regulation of PIN2 abundance
by ROP GTPases (Chen et al., 2012; Lin et al., 2012) that
have been independently shown to associate with NDs (Prior
et al., 2001, Sorek et al., 2007, 2010).
ABCB19 occurs in NDs that appear to be compositionally
different from the most common forms, and has been shown
to recruit PIN1 into, or maintain PIN1 within these domains
(Titapiwatanakun et al., 2009). The ABCB19/PIN1-ND is
formed during anterograde trafficking through the secretory
pathway (Yang et al., 2013). Short-term treatment with FEN
results in the initial accumulation of ABCB19 in a TGN/EE
compartment labelled by SYP61 before ultimate diversion to
the pre-vacuolar compartment (PVC) via vesicles marked by
VTI12 (Yang et al., 2013). This SYP61 compartment is part
of both exocytotic and endocytotic streams, and thus contains lipids and proteins from both pathways (Dettmer et al.,
2006). FRAP analysis of ABCB19–green fluorescent protein
(GFP) showed slow recovery of PM signals and enhanced
fluorescence recovery in FEN-treated SYP61 compartments,
Plant nanodomains and protein function | 1581
indicating that indeed exit of ABCB19–GFP from the SYP61
compartment is impaired in the presence of FEN. Thus, only
the last step in trafficking of ABCB19 through the secretory pathway is dependent on sterols. However, depletion
of SLs resulted in the accumulation of ABCB19–GFP in a
different compartment. Treatment with FB1 resulted in the
accumulation of ABCB19–GFP in the Golgi, and only a
fraction was able to reach the TGN and PM (Yang et al.,
2013). Similar results were observed in the SL biosynthesis
mutant tsc10a-2. These experiments indicate that SLs and
sterols assemble with proteins associated with this class of
NDs in discrete stages, with SL association preceding sterol
packing. Although these results differ from what had been
previously proposed for DRMs (Laloi et al., 2007), it is also
more consistent with the more recent recognition of GIPC
abundance in Arabidopsis PMs (Blaas and Humpf, 2013;
Markham et al., 2013).
ABCB4 is regularly detected in canonical DRMs (Borner
et al., 2005), and recently in SYP61 proteomics (Drakakaki
et al., 2012). ABCB4 is also reduced in tsc10a-2 and after
FB1 treatment, though to a lesser extent than ABCB19 (Yang
et al., 2013). ABCB4 localization to the PM is also sensitive
to very low concentrations of dimethylsulphoxide (DMSO;
0.05%), which is a common solvent for pharmacological
inhibitors, dyes, and growth regulators (Kubeš et al., 2012).
DMSO introduces water pores into lipid bilayers (Notman
et al., 2006), which could potentially disturb lipid–protein
interactions in ND-associated proteins. Such dissociation is
likely to contribute to the observed ND dissociation, subsequent internalization, and exposure of proteolytic cleavage
sites in ABCB4. For example, mammalian protein kinase C
is stable at the membrane when it interacts with acidic lipids.
In the presence of phosphatidylserine and diacylglycerol it is
destabilized, exposing a proteolytic site within the protein,
which ultimately leads to its degradation by Arg-C (Orr et al.,
1992).
A clear example of ND-dependent recruitment is seen
in ABA regulation of Arabidopsis SLAH3. SLAH3 is a
PM/ND-localized anion channel that is involved in ABA
signalling (Geiger et al., 2011). Without the indirect activation of SLAH3 by the presence of ABA, the protein is primarily found in detergent-soluble endomembranes (Demir
et al., 2013). SLAH3 activity depends strongly on its interaction with CPK21 at the PM, but this interaction is inhibited
in the absence of ABA through the binding of the signalling
phosphatase ABI1 to CPK21. The presence of ABA prevents
this interaction, as under these conditions ABI1 preferentially forms a complex with ABA-bound RCAR1/PYL9. In
the presence of ABA, the protein complex SLAH3/CPK21 is
associated with sterol- and SL-rich NDs (Fig. 2; Demir et al.,
2013). CPK21 localization to the ND is sterol dependent,
as treatment with MβCD releases it from the PM. SLAH3/
CPK21-ND formation occurs at the PM and not during
anterograde trafficking within the secretory pathway. This
association has been described as fully reversible and thus
appears to differ from ABCB19.
ABCG36/PEN3 is associated with multiple stress
responses including pathogen responses, suggesting that NDs
may function in a manner analogous to pathogen interactions observed in other kingdoms (Triantafilou et al., 2002;
Rogers et al., 2012). ABCG36/PEN3 is regarded as a component of pathogen-associated molecular patterns (PAMP)triggered immune response in Arabidopsis, and is activated
upon infection with the powdery mildew Blumeria graminis
(Underwood and Somerville, 2013). ABCG36/PEN3 exhibits
a uniform PM distribution in uninfected wild-type leaf cells
(Underwood and Somerville, 2013), and is also present within
the SYP61 compartment (Drakakaki et al., 2012). Upon
attempted fungal penetration, it forms distinct puncta at the
site of infection, where it accumulates in the extracellular
space within the papillae, into which it facilitates the transport
of antimicrobial metabolites. In the roots, ABCG36/PEN3 is
polar localized to the basal PM, designated as the outer polar
domain, which defines the interface between the plant and the
surrounding soil (Langowski et al., 2010). Notably, neither
FB1 (SL biosynthesis inhibitor) nor FEN (sterol biosynthesis
inhibitor) treatment appear to have a specific effect on its PM
localization (Yang et al., 2013).
Fig. 2. Schematic representation of the SLAH3/CPK21 and ABCB19/PIN1 membrane nanodomains. Left: SLAH3 is active in the presence of abscisic
acid (ABA). Binding of ABA to the RCAR/PYL9 receptor recruits ABI1 to the complex and blocks CPK21 interaction. This allows for the formation of the
SLAH3/CPK21 complex in sterol- and SL-rich nanodomains. Whether ABI1 shows preferential binding to either nanodomains (SL shown with yellow
head groups, sterols as small grey structures) or phospholipids (blue head groups) of the plasma membrane is not known. Centre: in the absence
of ABA, ABI1 binds to CPK21, and SLAH3/CPK21 complex formation is inhibited. Under these conditions, SLAH3 and CPK21 are mainly found in
detergent-soluble membranes. Red arrows indicate movement of CPK21 out of the nanodomains. Right: ABCB19/PIN1 interact in nanodomains with
the result of increased specificity and effectiveness of polarized auxin transport. Consistent identification of FLA2 and other GPI-anchored fasciclin-like
arabinogalactan proteins in nanodomains, and co-purification of FLA2 with ABCB19 suggest that a subclass of FLAs function in nanodomain–cell wall
interactions. Schematic representation of FLA2 interaction with the ND via the C-terminal GPI anchor is shown. Grey rectangle, ethanolamine; blue
circles, mannose; green circle, glucosamine; red lipid head group, inositol.
1582 | Tapken and Murphy
Inositol phosphoryl ceramide synthase (IPCS) converts
ceramide (SL) into inositol phosphorylceramide, which can
be subsequently converted to GIPC (Rennie et al., 2014).
IPCS was originally identified in an enhancer screen for
RESISTANCE TO POWDERY MILDEW8 (RPW8), which
confers the hypersensitive response to multiple powdery mildew pathogens (Xiao et al., 2001; Wang et al., 2008). In IPCSdefective mutants, the ceramide concentration is increased,
resulting in elevated salicylic acid accumulation and RPW8
expression, and ultimately to increased resistance to powdery
mildew (Wang et al., 2008). IPCS, like ABCB19, ABCB4, and
ABCG36/PEN3, was localized to the SYP61 compartment.
Thus, it appears to have a dual role in resistance to powdery
mildew, by controlling the amounts of the signalling lipid
ceramide through IPCS, and shuttling ABCG36/PEN3 to the
sites of infection. Together, this also focuses our attention on
the SYP61 compartment as an important component in ND
trafficking and formation. The presence of IPCS in this compartment raises the possibility that even after exit from the
Golgi, ND formation and restructuring of proteins could still
occur.
Do cell wall interactions stabilize NDs?
Although NDs are clearly shown to stabilize associated protein complexes at the PM, there is currently no evidence that
they function directly in polar trafficking events. However,
as is the case with polarized PIN1 auxin transporter (Feraru
et al., 2011), enzymatic removal and mutational disruption of
the cell wall results in a more diffuse and uniform distribution of ABCB auxin transporters at the PM. This suggests
that ND interactions with cell walls may occur via embedded
proteins with exoplasmic interaction structures. Interestingly,
the cell wall itself was shown to be sufficient for constraining
the movement of ND-partinioning minimal proteins (containing only the membrane-spanning domains; Martinière
et al., 2012). Two groups of proteins that consistently occur
in DRM fractions are the FLAs and jacalin lectin proteins.
FLAs are considered primary candidates for this function, as
they comprise a large family, are GPI anchored, and contain
arabinogalactan moieties that would be likely to interact with
cell wall carbohydrates (Elortza et al., 2003; Johnson et al.,
2003; Huang et al., 2013). However, such a function has not
yet been clearly demonstrated for specific NDs.
Conclusions
The role of ordered membrane domains in regulating the
trafficking and function of plant membrane proteins has
gradually moved from the realm of speculation to accepted
science. Studies of ND function in plants have been greatly
enhanced by elucidation of plant and mammalian pathogen
responses and the development of lipid-selective isolation
techniques. Recent elucidation of SL biosynthetic pathways,
development of a wide array of tools to explore PM–cell
wall interactions, and the adaptation of new techniques to
live imaging of plants have improved the characterization of
plant ND formation and function. Wholesale biochemical
isolation of DRMs has now been largely replaced by more
refined cell biological approaches that track candidate proteins as they are incorporated into and interact with other
proteins in sterol- and SL-rich domains. The ability to target
and modify ND-associated proteins selectively and visualize
them in planta with high-resolution microscopy techniques is
likely to clarify the precise functions of ND structures in the
near future.
Acknowledgements
This work was supported by the Department of Energy, Basic Energy
Sciences, grant no. DE-FG02-13ER16405 to ASM. General infrastructure support for the project was provided by the Maryland Agricultural
Experiment Station.
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