Journal of Cell Science 109, 777-787 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
JCS4117
777
Plant clathrin heavy chain: sequence analysis and restricted localisation in
growing pollen tubes
Hugh D. Blackbourn* and Antony P. Jackson
Biochemistry Department, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QW, UK
*Author for correspondence
SUMMARY
Clathrin-coated vesicles were isolated from soybean (Glycine
max L.) cells in suspension culture and their purity was
assessed using SDS-PAGE, peptide sequencing and electron
microscopy. Antibodies raised to these coated vesicles were
used to immunoscreen a soybean cDNA library in λgt11 and
isolate a partial clone of the clathrin heavy chain (HC) gene.
Full-length cDNA for soybean clathrin HC was deduced by
5′ and 3′ cDNA amplification. The cDNA encodes an amino
acid sequence of 1,700 residues, which is slightly larger than
rat clathrin HC and may account for the reduced mobility of
plant clathrin on SDS-PAGE. Insertion of these extra
residues is largely confined to the amino and carboxy termini.
Other domains within the heavy chain arms, including those
implicated in light chain binding and trimerisation, are relatively well conserved between eukaryotes. A computer
algorithm to determine α-helical coiled-coil structures
reveals that only one domain, aligning to residues 1,460-1,489
in rat clathrin HC, has a high probability for coiled-coil
structure in all five eukaryotic clathrin HC sequences. This
provides further evidence that the interaction between
clathrin heavy and light chains is mediated by three bundles
of coiled-coils near to the carboxy terminus. In analysing the
INTRODUCTION
Clathrin-coated vesicles are responsible for the selective
translocation of receptor-ligand complexes in a variety of
eukaryotic cells (Brodsky et al., 1991). At the plasma
membrane, macromolecules are internalised by receptormediated endocytosis, whilst at the trans-Golgi network a
distinct population of clathrin-coated vesicles is responsible for
the sorting and transport of lysosomal enzymes (Pearse and
Robinson, 1990). Our understanding of the structure of
clathrin-coated vesicles is largely derived from studies on
animal cells. These have revealed that the vesicle coat consists
of clathrin triskelions composed of clathrin heavy and light
chains, which assemble to form the polygonal lattice characteristic of these organelles. The clathrin light chains provide
the recognition site for coated vesicle disassembly by hsc70
and a variety of light chain isoforms have been identified
(Jackson and Parham, 1988). Underneath the clathrin shell lie
the adaptor complexes, which bind the selected receptor
role of plant clathrin in endocytotic trafficking, as against
trafficking from the Golgi apparatus to the vacuole, our
attention was focused on membrane recyling in tip-growing
pollen tubes. These rapidly growing cells are highly secretory
and require a high level of plasma membrane recycling to
maintain the tube tip architecture. Monoclonal antibodies to
plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled state,
suggesting a highly dynamic trafficking pathway. Immunofluorescence analysis of pollen tubes revealed that clathrin
localises to the plasma membrane at the apex of the pollen
tube tip, which is consistent with high levels of clathrinmediated membrane recycling. The use of these reagents in
conjunction with tip-growing pollen tubes has created a
unique opportunity to examine the basis for constitutive
endocytosis, so that the more complex question of receptormediated pathways in plants can also be assessed.
Key words: Clathrin coated vesicle, Endocytosis, Monoclonal
antibody, Heavy chain sequence, Pollen tube
molecules and cross-link them to clathrin. The adaptor
complex is a heterotetrameric structure composed of two 100110 kDa proteins, either α- and β-adaptin at the plasma
membrane, or γ- and β′-adaptin at the trans-Golgi network
(Pearse and Robinson, 1990). Also present are medium and
small chains of 50 kDa (µ2) and 17 kDa (σ2) at the plasma
membrane, or 47 kDa (µ1) and 19 kDa (σ1) at the trans-Golgi
network. A recent study has demonstrated that the medium
chain (µ2) of the plasma membrane-adaptor complex binds to
tyrosine-based signals of membrane proteins (Ohno et al.,
1995). This suggests that the medium chains of the adaptor
complex play a major role in dictating specificity, while the
remaining adaptor proteins create an appropriate environment
for receptor recruitment and clathrin binding.
There is at present only preliminary evidence for receptormediated endocytosis (RME) in plants (Horn et al., 1989) and
recent research on plant coated vesicles has concentrated on
their role in trafficking from the trans-Golgi network in developing pea cotyledons (Lin et al., 1992; Demmer et al., 1993).
778
H. D. Blackbourn and A. P. Jackson
It is nonetheless clear that clathrin also mediates membrane
retrieval at the plant plasma membrane (Fowke et al., 1991;
Mollenhauer et al., 1991). The use of cationised ferritin has
demonstrated that the internalisation of plasma membrane
follows essentially the same pathway as in animal cells. Thus,
coated pit formation is followed by vesicle budding, clathrin
uncoating, transport to the partially coated reticulum and from
there to the multivesicular body and the vacuole (Tanchak et
al., 1984; Fowke et al., 1991). In addition, the morphology of
plant and animal endocytotic vesicles appears to be almost
identical, although there are subtle differences in the Mr of
clathrin heavy chains and the length of the triskelion arms
(Coleman et al., 1987, 1988). While it is tempting to assume
that this reflects a common protein composition, the conservation of such vital components as clathrin heavy and light chains
and the adaptor complex components has yet to be demonstrated for plant coated vesicles. A deeper understanding of the
endocytotic process in plants will clearly require the same
detailed structural information that is available for animal cells.
Indeed, this may be the only effective means of assessing
whether RME occurs in plants, and in examining the molecular
basis of selectivity and specificity for coated pit assembly.
Progress in characterising clathrin-mediated endocytosis in
plants has been delayed by a variety of factors, including the
low recovery of coated vesicles, the barrier presented by the
plant cell wall and the lack of endocytotic markers, or crossreacting antibodies (see also Robinson and Hillmer, 1990).
Further progress will require a detailed structural analysis of
plant coated vesicles, the development of reagents to monitor
internalisation and selection of an appropriate plant cell in
which to perform this analysis. As a first step to resolving this
situation, we have isolated clathrin-coated vesicles from
soybean cells in suspension culture and analysed the principal
coat components. This has provided vital information on the
structure of plant clathrin and generated the necessary immunological and molecular probes to disect the pathway. The use of
these reagents has also allowed us to identify a suitable cell
type, the tip-growing pollen tube, which is amenable to further
genetic and biochemical analysis. We discuss the implications
of these results and possible avenues of further research.
MATERIALS AND METHODS
Plant material
Suspension cultured soybean (Glycine max L.) cells were grown in
400 ml of liquid B5 medium in 1 l flasks on a rotary shaker at 25°C
in continuous darkness. Anthers were collected 2 days after anthesis
from flowers of Lilium longiflorum (obtained from a local florists) and
air dried for 8 hours followed by 16 hours at −18°C. Pollen was
separated from the anthers with a brush and stored at −70°C until use.
Isolation of coated vesicles
Cell suspension cultures were resuspended in fresh media 24 hours
before the start of the experiment. The cells were harvested by filtration through 2 layers of muslin and resuspended in 2 volumes of
buffer A composed of 0.1 M Mes-NaOH, pH 6.5, 0.3 M sorbitol, 0.2%
BSA, 1 mM EGTA, 3 mM EDTA, 0.5 mM MgCl2, 0.02 % NaN3, 4
mM DTT, 1 mM PMSF, 1 µM pepstatin and 1 µg/ml leupeptin. The
suspension was homogenised using a bead beater for 1 minute per
load (15 second pulse; 45 second delay). The homogenate was centrifuged successively at 500 g for 5 minutes, 5,000 g for 10 minutes,
20,000 g for 30 minutes and 120,000 g for 1 hour. The post-microsomal pellets were resuspended in 4 ml buffer B (same as buffer A,
but without sorbitol), loaded onto linear gradients of 9% to 90% D2O
and centrifuged at 40,000 g for 30 minutes. The supernatant was
decanted, diluted twofold with buffer B and centrifuged at 120,000 g
for 1 hour. The pellets were resuspended in buffer B, layered onto a
linear gradient of 9% D2O/2% Ficoll to 90% D2O/25% Ficoll and centrifuged at 80,000 g for 16 hours. Successive fractions were recovered
from the gradient, diluted fourfold with buffer B and centrifuged at
120,000 g for 1 hour. The resulting pellets were resuspended in buffer
B and those fractions containing clathrin heavy chain (as determined
by SDS-PAGE) were pooled, made up to 5 ml, layered on to a 30 ml
gradient of 5% to 25% sucrose in buffer B and centrifuged at 100,000
g for 90 minutes. Fractions corresponding to 10% to 20% sucrose
were collected, diluted with 2 volumes of buffer B and centrifuged at
100,000 g for 60 minutes. The resulting clear pellet was resuspended
in a small volume of buffer B and stored at either 4°C, or at −70°C
until use.
Isolation of pig brain coated vesicles
Coated vesicles were isolated from freshly obtained pig brains by differential centrifugation and gel-filtration on Sephacryl S-1000 as
described (Cambell et al., 1984).
Polyclonal and monoclonal antibodies
Balb/c mice were immunised with sucrose-purified clathrin-coated
vesicles from soybean, using standard procedures (Galfre and Milstein,
1981). Spleen cells of mice showing an immune response were fused
with P3-NS1/1-Ag4-1 myeloma cells and screened for antibodies to
plant clathrin by western blotting. Following cloning at limiting
dilution, antibody was purified by affinity chromatography (HiTrap,
Pharmacia) and characterised using an antibody isotyping kit (Pierce).
The monoclonal antibody ‘4A8’, is an IgG1 immunoglobulin.
Gel electrophoresis and immunoblotting
Gel electrophoresis was carried out on 8% resolving gels using the
buffer systems of Laemmli (1970). Gels were either stained with
Coomassie Brilliant Blue R-250, or by silver staining using silver
stain plus (Bio-Rad). Transfer to Hybond-C (Amersham International)
was carried out in 20% methanol, 25 mM Tris, 192 mM glycine and
0.02% SDS at 100 V for 1 hour. After transfer membranes were
washed briefly in TBS and incubated in TBS containing 5% low fat
milk for 1 hour at room temperature. The blot was added to primary
antibody diluted in TBS containing 1% BSA and incubated overnight
at 4°C. The membrane was washed for 5 minutes each in TBS,
TBS+0.5 M NaCl, TBS+0.1% Triton X-100 and TBS. The blot was
then incubated with peroxidase-conjugated second antibody in
TBS+1% BSA for 1 hour at 4°C. Membranes were washed as before
and visualised using either a peroxidase staining kit (Vector Labs.) or
ECL (Amersham).
Isolation of a soybean clathrin HC clone
A λgt11 soybean stretch library (Clontech no. FL1062b) was
immunoscreened according to standard protocols (Sambrook et al.,
1989), using immune serum to soybean clathrin HC. Out of 350,000
plaques screened, a single immunoreactive plaque was detected and
the 3.2 kb insert excised by EcoRI digestion and subcloned into
M13mp18. Sequencing of both strands was performed by dideoxy
chain termination (Sanger et al., 1977) using a T7 sequencing kit
(Pharmacia) and 35S-dATP (Amerham International). The remaining
5′ and 3′ coding regions were identified by Marathon™ cDNA amplification (Clontech). Total RNA was isolated from freeze-dried
soybean cells using standard plant protocols (Sambrook et al., 1989)
and poly(A)+ RNA purified using a PolyATtract™ isolation kit
(Promega). Marathon™ cDNA synthesis and adaptor ligation was
performed as described by Chenchik et al. (1995), with the following
exceptions: second strand synthesis was allowed to proceed for 6
Plant clathrin heavy chain
hours at 16°C; following the addition of T4 DNA polymerase the
sample was incubated for 2 hours at 16°C and the adaptor ligated
cDNA was used directly in long distance PCR, as dilution in TricineEDTA buffer prevented priming. The complete cDNA of the soybean
clathrin HC was deduced using synthetic oligonucleotides to bridge
the PCR products. Automated sequencing of both strands of DNA was
by dye termination reaction.
DNA blots
Genomic DNA was isolated from soybean cells in suspension culture
according to the method of Dellaporta et al. (1983) and Southern
analysis was performed using the method of Sambrook et al. (1989).
Probes labelled with [32P]dCTP were prepared by random priming
DNA labelling (Boehringer-Mannheim). Hybridisations were at 42°C
in the presence of 50% formamide.
Sequence analysis
Sequences were compared using PILEUP (University of Wisconsin
Genetics Computer Group analysis software package) with a gap
weight of 3.0 and a gap length weight of 0.1. Probabilities of forming
coiled-coil structures were determined using the algorithm of Knight
and Kendrick-Jones (1993) based on the programme developed by
Lupas et al. (1991). Changes to the programmes’ parameters made it
necessary to repeat the analysis of the rat, Dictyostelium and yeast
clathrin HC sequences performed previously (Nathke et al., 1992),
while including the probability plots for soybean and Drosophila
clathrin HC sequences.
Detection of clathrin-coated vesicles in microsomal
fractions from pollen tubes
Pollen from L. longiflorum (10 mg pollen per ml of buffer) was
incubated in germination buffer (Kohno et al., 1990) on a rotator for
90 minutes at 30°C. Germinated pollen was collected by centrifugation at 600 g for 2 minutes and resuspended in buffer B. Pollen tubes
were homogenised using a Potter homogeniser and centrifuged at 600
g for 1 minute to remove cell debris. The supernatant was re-centrifuged at 14,000 g for 1 minutes, the supernatant decanted and centrifuged at 100,000 g for 20 minutes at 4°C. The resulting pellet was
resuspended in a small volume of buffer B and analysed by SDSPAGE.
The ratio of assembled/disassembled clathrin during
pollen tube growth
Microsomal extracts were prepared as above using a buffer composed
of 10 mM Hepes (pH 7.4), 150 mM NaCl, 1 mM EGTA, 0.5 mM
MgCl2, 0.2% Triton X-100, 4 mM DTT and 0.2% azide (Acton et al.,
1993). Following centrifugation at 100,000 g, supernatants were
decanted and the pellets resuspended in sample buffer. TCA was
added to the supernatants to a final concentration of 10% and the
samples were left overnight at 4°C. The TCA precipitate was collected
by centrifugation at 16,000 g for 10 minutes at 4°C and washed in
cold 100% acetone for 30 minutes at 4°C. Following centrifugation
at 16,000 g for 10 minutes the pellets were resuspended in sample
buffer and aliquots of supernatant and pellet fractions were resolved
on SDS-PAGE. The proportion of clathrin HC in each fraction was
analysed by probing western blots with the monoclonal antibody 4A8.
Immunofluorescence confocal microscopy
Pollen tubes were germinated as above and allowed to settle for 5
minutes. The growth medium was decanted and the pollen tubes
resuspended in 100 mM Pipes buffer (pH 7.0) containing 4%
formaldehyde and 4 mM EGTA. The samples were left to fix for 1
hour at 20°C. The following solution changes were accomplished by
centrifugation at 600 g for 1 minute. After a brief wash in PBS, pollen
tubes were permeabilised by addition of PBS containing 0.5% Triton
X-100 for 10 minutes. The samples were briefly washed in PBS,
resuspended in blocking buffer (PBS containing 5% foetal calf serum,
779
0.1% Triton X-100, 0.02% SDS, 5% horse serum and 0.02% azide)
and left for 1 hour. Pollen tubes were then incubated with 1o antibody
in blocking buffer. Following three 5-minute washes in PBS, pollen
tubes were resuspended in blocking buffer containing FITC-conjugate
of goat anti-mouse IgG and left for 1 hour. The pollen tubes were
given three 5-minute washes in PBS, one 5-minute wash in distilled
water and allowed to air-dry onto the surface of a microscope slide.
Pollen tubes were mounted in 0.2 M Tris-HCl (pH 8.3), 85% glycerol
and 3% n-propyl gallate, and viewed using a Bio-Rad MRC-1000
confocal microscope. Control experiments included the use of preimmune sera, the omission of 1o and 2o antibodies and labelling with
a monoclonal antibody to α-tubulin.
RESULTS
Purification and analysis of plant coated vesicles
We elected to isolate clathrin-coated vesicles from soybean
cells in suspension culture, as these are a recognised source of
endocytotic coated vesicles (Coleman et al., 1988). The
protocol employed was adapted from that developed for
soybean protoplasts (Mersey et al., 1985) and included EDTA
in the extraction buffers to avoid contamination by ribosomes,
and azide to inhibit ATP-dependent uncoating. Protein assays
revealed that the recovery of these highly purified coated
vesicles was relatively low, with 0.2 µg protein g−1 FW. This
value is only 3% to 5% of that reported for developing pea
cotyledons and brain tissue, respectively (Harley and Beevers,
1989). However, it appears that coated vesicles from pea
cotyledons (in which Golgi-trafficking predominates) are contaminated by phytoferritin, which may lead to an overestimation of their recovery (Hoh and Robinson, 1993). There is no
equivalent 28 kDa contaminant in the sucrose-purified coated
vesicles from soybean.
The profile of highly purified coated vesicles on SDS-PAGE
is strikingly similar to that of animal coated vesicles, with a
dominant band of 185 kDa and relatively few other bands, of
which those around 110 kDa and a doublet of 50-54 kDa are
the most obvious (Fig 1[i]A+B). Immunoblotting confirmed
that the 50-54 kDa doublet is tubulin: this is not unexpected as
tubulin co-purifies with mammalian clathrin-coated vesicles.
Silver staining reveals that other polypeptides are also present
with bands at 130-140 kDa, a doublet at 110 kDa and another
at 90 kDa (Fig. 1[i]C). Silver staining also reveals the presence
of a number of lower molecular mass polypeptides, but it is
not clear which, if any, are clathrin light chains. The soybean
coated vesicle preparations were judged to be free from contamination following ultrastructural analysis of negatively
stained specimens (Fig. 1[ii]).
Identification and sequence analysis of soybean
clathrin HC
The amino terminus of soybean clathrin heavy chain is
blocked, but internal amino acid sequence was obtained by
partial digestion with V8 protease (Cleveland, 1983) and
peptide sequencing (see legend to Fig. 3). However, we
reasoned that the highly degenerate nature of oligonucleotides
generated to these sequences would prohibit stringent library
screening, and were therefore prompted to raise antibodies to
plant clathrin, using sucrose-purified clathrin-coated vesicles
from soybean as antigen. A polyclonal antiserum with high
affinity for plant clathrin HC on western blots (see Fig. 6a),
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(i)
H. D. Blackbourn and A. P. Jackson
(ii)
Fig. 1. A comparison between coated vesicles from soybean and pig
brain. (i) Coated vesicles from pig brain (A) and coated vesicles
from soybean purified on a sucrose gradient (B), were resolved on
SDS-PAGE and stained with Coomassie Brilliant Blue. Less intensly
staining bands in the soybean samples were revealed by silver
staining (C). (ii) Negatively stained samples of soybean coated
vesicles examined by electron microscopy. Molecular mass (kDa) is
given on the left.
was then used to immunoscreen a soybean cDNA library in
λgt11. Screening of up to 350,000 plaques gave a single
positive plaque containing an insert of 3.2 kb. Sequencing
revealed that the 3.2 kb fragment was approximately 60%
identical to rat clathrin and the translated sequence spanned
amino acid residues 275-1346 (Kirchhausen et al., 1987).
Further screening using 32P-labelled cDNA probes of this
fragment failed to identify other homologous sequences in the
library used. The remaining heavy chain sequences were
therefore cloned by PCR using oligonucleotides raised to
sequences mapping to the 5′ and 3′ ends in conjunction with
Marathon™ cDNA ampification. The resulting cDNA includes
an AUG at the start that resides within an authentic translation
sequence (Grierson and Covey, 1988) and provides an open
reading frame of 5,100 bp. The nucleotide sequence provides
a corresponding translated amino acid sequence of 1,700
residues. The calculated size of soybean clathrin HC (193,353
Da) is slightly larger than that calculated for rat clathrin HC
(191,597 Da) and may explain the reduced mobility of soybean
clathrin HC on SDS-PAGE.
DNA Southern blot analysis at high stringency, using a
cDNA probe from the coding region (bases 2,022-3,876), gave
two bands in soybean genomic DNA restricted with either
EcoRI or BamHI (Fig. 2). The intensity and size of these bands
are consistent with the position of restriction sites within this
region and the proportion of template available for random
priming either side of the restriction site. This suggests that
only one gene codes for clathrin and there are no pseudogenes,
or highly homologous genes, in the soybean genome.
The previously determined peptide sequence for V8 protease
digests of soybean clathrin HC confirmed that the translated
amino acid sequence is a legitimate soybean gene product (Fig.
3). The amino acid sequence of the soybean clathrin HC is
most closely related to the other eukaryotic clathrin HC
sequences in the following order: rat (56% identity; 75% sim-
Fig. 2. Southern blot
analysis. Genomic DNA for
cell suspension-cultured
soybean cells was digested
with EcoRI (lane 1), or
BamHI (lane 2), resolved on
0.7% agarose at 10 µg per
lane and capillary transferred
to nylon membrane. The blot
was probed with clathrin HC
cDNA labelled by random
priming across nucleotides
2,022-3,876 of the coding
region. Size markers to the
left of the blot are in kb.
ilarity), Drosophila (56% identity; 74% similarity), Dictyostelium (54% identity; 74% similarity) and yeast (44%
identity; 68% similarity). Multiple sequence alignments reveal
that the greatest degree of divergence is confined to the aminoterminal and carboxy-terminal residues. There are a number of
major gaps introduced into the first 250 residues of the eukaryotic sequences, some of which are unique to soybean HC. The
fact that extra residues are accommodated in the N-terminal
domain of soybean HC may satisfy the point that structural
restraints are less vital in these domains than in the extended
arms of the HC, which make intimate contact with each other
and with clathrin light chains (Lemmon et al., 1991; Nathke et
al., 1992).
There is evidence that the light chain binding and trimerisation sites of mammalian HC lie adjacent to each other,
spanning residues 1,460-1,489 and 1,490-1,587, respectively
(Nathke et al., 1992). Multiple sequence alignments reveal that
both these sites are well conserved in soybean clathrin HC. The
C-terminal domain (residues 1,590-1,675 in mammals) may
form the globular protrusion on top of triskelia at the vertex
(Nathke et al., 1992). It is evident that the residues at the
extreme C termini are very poorly conserved between species.
Analysis of a C-terminal mutant of yeast HC has already
demonstrated that this globular protrusion plays no essential
role in triskelia assembly or clathrin function (Lemmon et al.,
1991).
An analysis of clathrin HC and light chain sequences
suggests that there is a high probability of their interacting via
Fig. 3. Alignment of the clathrin HC amino acid sequences from
soybean (G.m.), Drosophila (D.m.; Bazinet et al., 1993), rat (R.n.;
Kirchhausen et al., 1987), Dictyostelium (D.d.; O’Halloran and
Anderson, 1992) and yeast (S.c.; Lemmon et al., 1991). The
nucleotide sequence for soybean clathrin HC will appear in the
EMBL, GenBank and DDBJ databases under the accession number
U42608. Identical residues in all five sequences are indicated by a
dash (-). Gaps in the alignment introduced by PILEUP are shown as
a period (.). The position of peptide sequence obtained from V8
protease digests of soybean clathrin HC (APQXXVVIIDM and
NLVTFPNVADAILANGMFS) are shown underlined in the soybean
sequence.
Plant clathrin heavy chain
781
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H. D. Blackbourn and A. P. Jackson
Fig. 5. Monoclonal antibodies to plant clathrin HC and their use to
assess the proportion of assembled clathrin in pollen tubes of L.
longiflorum. (A) Microsomal extracts from pollen tubes of lilium (L)
and soybean cells (S) equivalent to 1 mg of pollen and 100 mg of
soybean cells, stained with Amido Black (left-hand panel) and
immunoblot challenged with monoclonal antibody 4A8 (right-hand
panel). (B) The ratio of assembled to unassembled clathrin
triskelions was examined at various times after germination in vitro.
Equal samples of the pellet (p) and supernatant (s) fractions were
resolved on SDS-PAGE and the proportion of clathrin HC in each
fraction was determined by immunoblotting with the monoclonal
antibody 4A8.
Fig. 4. Analysis of α-helical coiled-coils in clathrin HC sequences.
The five eukaryotic clathrin HC sequences (A is soybean; B is
Drosophila; C is rat; D is Dictyostelium and E is yeast) were
submitted to a computer algorithm to determine the probability of
their forming coiled-coil structures (Knight and Kendrick-Jones,
1993), using a window size of 28. This is an updated version of the
programme developed by Lupas et al. (1991), in which the residue
frequency within the seven positions of a heptad repeat was used to
generate a score for each residue in the sequence. The resulting
scores were then compared with known globular or coiled-coilcontaining proteins to determine the probability of the sequence
forming a coiled-coil. Previous analysis of clathrin HC sequences
(Nathke et al., 1992), had identified amino acids aligning to rat
residues 1,151-1,178 (low probability) and 1,460-1,489 (high
probability) as possible α-helical coiled-coils.
α-helical coiled-coils (Nathke et al., 1992). This was established using a computer programme to compare residues and
their flanking sequences with residues in known coiled-coil
structures (Lupas et al., 1991). Equivalent analysis of soybean
HC, using a modified form of this programme (Knight et al.,
1993), gives a high probability of α-helical coiled-coil from
residues 1,475-1,502 (Fig. 4). These residues align to the
equivalent domain of residues 1,460-1,489 in the rat and other
eukaryotic sequences. This is the only domain that gives a consistently high probability of coiled-coil structures in all five
sequences. As previously noted the weak prediction of a heptad
repeat pattern for residues 1,151-1,178 in mammalian HC
(Kirchhausen et al., 1987) is not shared by yeast HC (Nathke
et al., 1992) and the heptad pattern for soybean HC is also
weak. The coiled-coil prediction near to the C-terminal domain
of soybean HC (residues 1,617-1,644), which may form the
protrusion near to the vertex of the triskelia (Nathke et al.,
1992), is more pronounced than the equivalent region in rat
HC, but similar to that calculated for dictyostelium HC.
Clathrin-mediated membrane recycling in pollen
tubes
The prominent band of 185 kDa apparent in microsomal
extracts from pollen tubes of L. longiflorum on SDS-PAGE
was blotted and probed with monoclonal antibody 4A8, raised
to soybean clathrin HC (Fig. 5A). This revealed that the
procedure used to isolate microsomal extracts results in greater
recovery of both total protein and clathrin from pollen tubes of
L. longiflorum than from an equivalent sample of soybean
cells. This may merely reflect differences in the degree of vacuolation between the two cell types, but it is a useful indication of the suitability of pollen tubes for coated vesicle
analysis. It is also consistent with the high levels of clathrinmediated endocytosis reported for similarly highly secretory
plant cells (Steer, 1988). The monoclonal antibody used specifically recognises clathrin HC in plants and we have also
employed it to qualitatively assess the proportion of clathrin
that is present in either the assembled, or the unassembled, pool
of clathrin. This revealed that a very high proportion of the
clathrin HC in growing pollen tubes is present in an assembled
state (Fig. 5B). This compares with mammalian cells in which
30-35% of the clathrin heavy chain is assembled in cells with
low endocytotic activity and 90% in cells that are endocytotically and/or exocytotically active (Goud et al., 1985).
Plant clathrin heavy chain
783
Fig. 6. Immunolocalisation
of clathrin in pollen tubes.
(A) Immunoblot of crude
microsomal extracts from
pollen tubes of L.
longiflorum to demonstrate
the specificity of the
immune serum for clathrin
HC. The distribution of
clathrin HC was assesed in
pollen tubes fixed with
formaldehyde and
permeabilised with Triton
X-100.
(B) Immunofluorescent
staining in the tip region
revealed in a composite
image of a pollen tube, and
(C) the staining pattern of
an optical section 5 µm
below the upper plane of
the pollen tube. (D) A
similar pattern was
observed at the apex of all
optically sectioned pollen
tubes. (E) There was no
immunoflourescent
staining of pollen tubes for
any of the control
incubations, including pre-immune serum, shown here under epifluorescence illumination to reveal the position of the tube. The clathrin HC
immune serum was detected using FITC- or peroxidase-labelled goat anti-mouse serum. Bars, 10 µm.
The distribution of clathrin-coated structures was examined
in formaldehyde-fixed and detergent-permeabilised pollen
tubes using antibodies raised to soybean clathrin heavy chain.
The monoclonal antibodies that we had raised to soybean
clathrin HC recognise plant clathrin HC on SDS-PAGE and
native protein dot-blotted on to membrane, but failed to crossreact with clathrin in formaldehyde-fixed cells. However, the
polyclonal antiserum used in the immunoscreening protocol
for clathrin HC was effective at revealing the distribution of
clathrin in pollen. Western blots of crude extracts from pollen
tubes of Lilium revealed that the polyclonal mouse antiserum
specifically recognises clathrin HC (Fig. 6A). In cells, the distribution of clathrin HC was restricted to the tube tip apex, with
an intense signal extending up to 10 µm from the tip (Fig. 6B).
Optical sectioning through the upper face of the pollen tube
revealed that this signal was concentrated at the plasma
membrane of the tube wall, the logical site for clathrin-coated
pits implicated in membrane recycling in pollen tubes (Fig.
6C,D). The intense staining at the tube apex differs from the
punctate staining pattern reported for receptor-mediated
pathways in endocytotic mammalian cells (Brodsky, 1988).
However, in the pollen tube both exocytosis and membrane
recycling appear to be restricted to a zone near the apex, while
receptor-mediated endocytosis in other eukaryotes occurs in
clusters of coated pits dispersed over the surface of the plasma
membrane. There is no evidence of a requirement for clathrinmediated vacuolar trafficking in the vegetative cell of pollen
tubes (Noguchi, 1990), but we cannot rule out the possibility
that such a mechanism exists in the generative cell as aut-
oflourescence of the generative cell prevented this analysis
(results not shown). However, the contribution that clathrin
makes to trafficking in the generative cell is likely to be small
compared to that at the tip apex, given that there are relatively
few organelles within the generative cell cytoplasm. No fluorescence (other than autofluorescence of the germinative cell)
was detected in pollen tubes incubated with pre-immune sera
(Fig. 6E), with secondary antibodies alone or with no antibodies.
DISCUSSION
The results presented here further clarify the structure of plant
clathrin HC and provide evidence that endocytotic membrane
recycling in pollen tubes proceeds through a clathrin-mediated
pathway. The cloning and sequencing of the soybean clathrin
HC are the first departures from the traditional biochemical
analysis of coated vesicle trafficking in plants. A comparison
of the clathrin HC sequence from plants with those of other
eukaryotes reveals interesting insights into those domains that
are conserved or diverged during speciation. The increased
number of residues in soybean HC provides a plausible explanation for the higher Mr of plant clathrin on SDS-PAGE,
although the relationship between sequence size and gel
mobility does not always apply, as in the case of yeast clathrin
HC (Lemmon et al., 1991). Interestingly, plant heavy chains
are reported to be approximately 60 nm in length, while those
of mammals are 45-55 nm in length (Coleman et al., 1987).
784
H. D. Blackbourn and A. P. Jackson
These differences in arm length do not appear to alter the
overall dimensions of assembled polygons and hexagons in the
coat structure (Coleman et al., 1987). These authors predicted
that up to 80-90 amino acids arranged as an α-helix would be
required to account for this size difference. We find no direct
evidence for this, the 25 extra residues in soybean HC relative
to rat HC, are largely accommodated in the terminal domains,
which form the large globular protrusions and appear to make
little contribution to arm length. Interestingly, gaps that appear
in the alignment of soybean with other clathrin HC sequences
occur in the region preceding the flexible linker (residues 479523 in rat), which allows the globular N-terminal domain to
project inwards and make contact with the adaptor complex
(Kirchhausen et al., 1987). It is possible that these differences
in soybean clathrin HC terminal sequences may reflect subtle
changes in the association of plant triskelia with the underlying adaptor complex.
Two sequence domains of great structural importance are the
trimerisation domain and the light chain binding domain. The
unique trimeric structure of clathrin is mediated by noncovalent association of HC monomers and the sequence of rat
clathrin HC initially suggested that the proline-rich region of
the carboxy terminus (residues 1,631-1,675) could be responsible for this interaction (Kirchhausen et al., 1987). Subsequent
analysis of a mutant yeast clathrin HC gene lacking this
domain revealed that clathrin was present as trimers and that
the mutant was able to complement clathrin HC-deficient yeast
(Lemmon et al., 1991). A revised model of the clathrin triskelion now suggests that residues 1,488-1,587 are responsible for
trimerisation and residues 1,438-1,481 include sequences
required for light chain binding (Nathke et al., 1992). This
model was based on peptide mapping, immuno-electron
microscopy and computer predictions of coiled-coil structures.
The alignments and coiled-coil calculations for soybean HC
also highlight the conservation and importance of residues
1,475-1,502. This domain aligns to the sequence 1,460-1,489
in rat HC and is the only coiled-coil site in all five eukaryotic
species. The importance of the weakly predicted pattern of
heptad repeats spanning residues 1,107-1,184 in rat clathrin
HC (Kirchhausen et al., 1987) is further reduced by this comparison. This makes it unlikely that the light chains associate
with heavy chain trimers in ‘bundles’ of four coiled-coils as
envisaged for mammalian triskelia. The coiled-coil prediction
also has a bearing on the identification of plant clathrin light
chain homologs, which appear to be poorly conserved between
species, and so may be difficult to identify from sequence comparisons alone. In view of the evidence for coiled-coil interactions between clathrin HC and clathrin light chains, one can
hypothesise that authentic plant clathrin light chains will have
a similar probability of forming a coiled-coil structure.
Endocytotic recycling in plants appears to be closely linked
to secretory activity, while evidence of RME remains largely
circumstantial and mostly concerns the uptake of fungal
elicitors (Low and Chandra, 1994). In order to develop a model
of plasma membrane recycling, we have focused on endocytotic recycling during the growth of pollen tubes. These exhibit
rapid growth rates (up to 1 cm h−1) and have highly developed
trafficking pathways. Previous analysis of pollen biology has
revealed that vesicle fusion with the plasma membrane results
in excess membrane at the tube tip, up to 80% of which may
be recycled (Steer, 1988), and the uptake of FITC-labelled
dextran demonstrates that endocytosis is particularly active at
the tube tip (O’Driscoll et al., 1993). Our analysis of pollen
tubes of Lilium revealed that coated vesicles are relatively
abundant and that almost all of the clathrin HC present is in an
assembled state during tube growth. Our immunofluorescence
analysis demonstrated that clathrin is concentrated at the
extreme apex of the tube tip. This distribution corresponds to
the site of secretory vesicle fusion and the point of entry of
FITC-labelled dextran by endocytosis, and fits well with the
known organisation of pollen tubes, which has been examined
following both chemical fixation, and rapid freeze fixation and
freeze substitution. In chemically fixed pollen tubes there
appear to be a number of distict zones, in the first 5 µm of the
tip there is a cap of tightly packed vesicles, this is followed by
a zone rich in ER and mitochondria and then another subapical
zone approximately 10-15 µm from the tip where Golgi and
other organelles appear (Picton and Steer, 1981; Steer and
Steer, 1990). In freeze-substituted pollen tubes this pattern is
repeated, but a number of randomly orientated elements of ER
can also be seen at the vesicle-rich tip apex (Lancelle et al.,
1987; Lancelle and Hepler, 1992). There is evidence that two
populations of smooth vesicles are present at the pollen tube
tip, the secretory vesicles of 300 nm diameter and unidentified
vesicles of 50 nm diameter (Cresti and Tiezzi, 1990). The high
levels of assembled clathrin present in pollen tubes are consistent with rapid rates of uncoating and recycling to the
plasma membrane, so the 50 nm diameter vesicles may be
uncoated vesicles trafficking back from the apex. The fate of
this re-internalised membrane is not clear. Perhaps the
membrane structures reported to be present at the tip apex in
freeze-substituted pollen tubes (Lancelle and Hepler, 1992)
serve as an early endosomal compartment for recycling. These
results suggest that clathrin is dedicated to selective membrane
recycling following secretory vesicle fusion in pollen tubes.
This role may be similar to that played by clathrin during
synaptic vesicle recycling in nerve terminals, where there is
evidence that synaptic vesicle membrane proteins recruit AP2 to the plasma membrane (Maycox et al., 1992; Zhang et al.,
1994). An interesting question that arises from these observations on pollen tubes is: what directs coated pits to assemble
at this precise position on the plasma membrane? Currently,
there is no information on how plant clathrin is recruited to the
plasma membrane, but since adaptins are part of the coat
structure it is likely that as in animal cells they interact with
specific membrane proteins. The vegetative pollen cell is an
ideal system in which to examine the biochemistry of this
process. Techniques have already been developed to isolate
secretory vesicles and plasma membrane-rich fractions from
pollen tubes (Van der Woude et al., 1971), and antibodies to
clathrin HC and β-adaptin can be used to immunoprecipiate
coated vesicles, so revealing those proteins that are selectively
internalised. A similar protocol has been used to demonstrate
that immunoprecipitation of the EGF receptor of A-431 cells
results in recovery of almost half of the available adaptin pool
(Sorkin and Carpenter, 1993). The isolation of vegetative cellspecific pollen promoters (Twell, 1992) has also created the
means to peturb clathrin levels within the growing pollen tube,
using antisense constructs to the heavy chain and N-terminal
mutants to create a variety of clathrin-deficient phenotypes.
In conclusion, antibodies and molecular probes to clathrin
are now available and we have described an interesting plant
Plant clathrin heavy chain
cell in which clathrin plays an important role in endocytotic
trafficking. This provides an exciting new dimension to vesicle
trafficking and a unique opportunity to advance our level of
understanding of this process in plants. Isolation of a plant
clathrin HC cDNA sequence also creates an opportunity to
assess functional aspects of clathrin HC structure. Since plant
clathrin HC is not recognised by antibodies to mammalian HC
available to us, expression of deletion constructs of soybean
HC in rat PC12 cells would allow us to discriminate between
plant and animal clathrin HC in assembled triskelions and so
deduce trimerisation and light chain binding domains. A
similar analysis could also explore the interaction between Nterminal sequences and the underlying adaptor complex.
This research was supported by an Agricultural and Food Research
Council grant PG008/065 and an award from the Royal Society
Research Grant Scheme for monoclonal antibody production.
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(Received 16 October 1995 - Accepted 2 January 1996)