From the
The major Euglena thylakoid protein, the light
harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII)
is synthesized in the cytoplasm as a polyprotein precursor composed of
a 141 amino acid presequence containing a signal peptide domain
followed by eight mature LHCPIIs covalently linked by a decapeptide. To
determine the transport route from cytoplasm to chloroplast and the
site of polyprotein processing, Euglena was pulse labeled with
[
Transport of proteins into the ER
In contrast to
protein transport into the ER, transport of proteins into chloroplasts
is post-translational. Chloroplast proteins are synthesized in the
cytoplasm as soluble precursors containing a N-terminal extension, the
transit peptide, that interacts post-translationally with envelope
receptors
(8) . The transit peptide functions as a stroma
targeting signal
(9) and additional targeting information is
required for localization within the chloroplast. LHCPII, the major
thylakoid protein, spans the membrane three times, has a stromal
targeting transit sequence that is not required for thylakoid
integration
(9, 10) , and the mature protein in a soluble
form is integrated into the thylakoid
(11) . Integration requires
all three membrane-spanning
domains
(12, 13, 14) .
The pLHCPIIs of
Euglena, a unicellular protist, are four proteins of 207, 161,
122, and 110 kDa encoded by 6.6- and 9.5-kilobase
mRNAs
(15, 16) . The sequence of two genomic clones
indicates that the pLHCPIIs are polyproteins composed of multiple
copies of LHCPII covalently joined by a decapeptide
linker
(16, 17) . Individual Euglena LHCPIIs are
approximately 65% homologous to higher plant LHCPIIs containing the
three hydrophobic membrane-spanning thylakoid insertion
domains
(16) . Euglena pLHCPII is synthesized on
membrane-bound polysomes
(18) as found for proteins translocated
into the ER rather than on free ribosomes as found for higher plant and
green algal pLHCPIIs. Euglena pLHCPII contains a
141-amino-acid N-terminal extension the first 35 amino acids of which
have the characteristic features of a signal peptide
(19) .
Euglena pLHCPII is co-translationally inserted in vitro into canine microsomes and the signal peptide removed
(19) .
During times of active LHCPII synthesis, immunogold electron microscopy
localizes Euglena LHCPII to the Golgi apparatus while at times
when LHCPII is not being synthesized, an immunoreaction is only seen in
the chloroplast
(20, 21, 22) . In contrast to
higher plant and green algal pLHCPIIs which are imported as soluble
precursors directly into chloroplasts, Euglena pLHCPII appears
to be co-translationally transported into the ER and transferred to the
Golgi apparatus prior to chloroplast localization.
The ER and Golgi
apparatus are the site of endoproteolytic processing of many
proproteins and polyprotein
precursors
(23, 24, 25, 26) suggesting
that pLHCPII polyprotein processing occurs in the ER or Golgi apparatus
prior to chloroplast localization. Pulse-chase intracellular
localization experiments were performed in order to delineate the
intracellular route of pLHCPII transport to the chloroplast and
identify the site of pLHCPII polyprotein processing. These experiments
provide the first direct in vivo demonstration of the
transport of Euglena pLHCPII from the ER to the Golgi
apparatus prior to chloroplast localization. pLHCPII is transported as
a membrane-bound polyprotein precursor whose endoproteolytic conversion
into individual LHCPIIs occurs during or rapidly after chloroplast
import. A brief report of this work has appeared
(27) .
Cells (10 ml)
exposed to light for 24 h were pulse-labeled with 600 µCi/ml of
carrier free H
The
clarified cell-free homogenate (1.5 ml) was loaded onto 12-ml gradients
consisting of a 2-ml 20% (w/w) sucrose step on top of a 25-50%
(w/w) linear sucrose gradient formed over a 0.5-ml cushion of 55% (w/w)
sucrose. Gradients were centrifuged at 100,000
Protease protection assays were performed
by incubating the 3,000
Organelle-specific marker enzyme distributions were used to
determine the location of specific subcellular organelles on isopycnic
sucrose density gradients. Chlorophyll, a marker for thylakoid
membranes and intact chloroplasts, formed two peaks between fractions
15-18 and 19-23 (Fig. 1). The small subunit of
ribulose bisphosphate carboxylase (SSU), a soluble chloroplast
protein
(38) , formed a peak coincident with the chlorophyll peak
in fractions 19-23 (Fig. 1) localizing intact chloroplasts
to this region. SSU was not found in the low density chlorophyll peak,
fractions 15-18 (Fig. 1), indicating that these fractions
contain thylakoid membranes. The SSU peak in fractions 1-5 at the
top (Fig. 1) of the gradient is protein released from broken
chloroplasts.
Glucose-6-phosphatase, localized exclusively in the Euglena ER
(38) , formed a single peak in fractions 3-6
(Fig. 1). Rotenone-insensitive NADH cytochrome c reductase, localized in both the Euglena ER and outer
mitochondrial membrane (42), formed a peak in fractions 3-6
(Fig. 1) confirming that ER membranes are localized to this
region of the gradient. The glucose-6-phosphatase, NADH cytochrome
c reductase ER peak was resolved, albeit poorly, from the peak
of solubilized SSU remaining at the top of the gradient (Fig. 1).
The major peak of rotenone-insensitive NADH cytochrome c reductase was in fractions 20-23 (Fig. 1). Succinate
dehydrogenase,
Large differences exist in the amount
of pLHCPII and LHCPII found in each gradient fraction (Fig. 2).
These differences exceed the linear range of fluorographic film
response. The fluorographic image therefore does not provide a
quantitative visual representation of the amount of pLHCPII and LHCPII
in subcellular organelles. To overcome this problem, storage
phosphorimaging which has a large linear response range was used to
accurately measure the amounts of pLHCPII and LHCPII in each fraction.
The SDS gels used for the fluorographs presented in Fig. 2were
scanned with a PhosphorImager. pLHCPII levels are defined as the sum of
the image intensity for the 207-, 161-, 122-, and 110-kDa pLHCPIIs
since the distribution of each of the four major pLHCPIIs was identical
to the total pLHCPII distribution. LHCPII levels are defined as the sum
of the image intensity for the 26- and 27-kDa LHCPIIs. The total
pLHCPII was present but did not form a distinct peak in thylakoids
and chloroplasts ( Fig. 2and Fig. 3). This material could
either represent thylakoid and chloroplast localized pLHCPII or ER and
Golgi localized pLHCPII that associates with chloroplast membranes
during subcellular fractionation. Chlorophyll provides a direct measure
of the amount of chloroplast membranes in each gradient fraction from
the 10-min pulse (Fig. 3A). If pLHCPII was transported
into chloroplasts and accumulated in thylakoids prior to processing,
the amount of pLHCPII in chloroplasts and thylakoids would be
proportional to the amount of chlorophyll recovered in the respective
fractions. Chloroplasts from the 10-min pulse-labeled sample, fractions
19-22, contained more chlorophyll than thylakoids, fractions
15-18 (Fig. 3A). Chloroplasts, however, contained
significantly less pLHCPII than thylakoids (Fig. 3A).
The pLHCPII in the thylakoid region of the gradient is probably Golgi
localized material that has been trapped by chloroplast membranes
during centrifugation and not pLHCPII accumulating in chloroplasts and
thylakoids prior to processing.
The fraction of total
immunoprecipitate recovered as pLHCPII in the ER, fractions 3-6,
decreased and the fraction in the Golgi apparatus, fractions
10-18, increased during a 20 or 40 min chase (Fig. 2, B and C, and 3 B and C). Partially
processed pLHCPIIs were not detected on the gradient (Fig. 2,
B and C). At the end of a 10-min pulse, 20% of
pLHCPII was in the ER and 70% in the Golgi apparatus. After a 20-min
chase, only 12% of pLHCPII was in the ER while 85% was in the Golgi
apparatus. By the end of the 40-min chase, 12% of pLHCPII was ER
localized and 70% was Golgi localized. Although the distribution of
pLHCPII between the ER and Golgi varied from experiment to experiment,
the fraction of ER localized pLHCPII was always significantly higher at
the end of a 10-min pulse than after a 20- or 40-min chase.
The
subcellular localization of LHCPII was distinctly different from
pLHCPII ( Fig. 2and Fig. 3). LHCPII was not present in the
ER, fractions 3-6. The disappearance of pLHCPII during a 20- or
40-min chase was associated with the appearance of LHCPII in
thylakoids, fractions 15-18, and chloroplasts, fractions
19-22 (Fig. 3, B and C). The LHCPII
thylakoid peak was clearly distinct from but overlapped the pLHCPII
Golgi peak (Fig. 2, B and C and 3, B and C). The absence of LHCPII from the ER and most
pLHCPII containing Golgi fractions suggests that pLHCPII is transferred
as a polyprotein from the ER and Golgi apparatus to the chloroplast
where during or rapidly after chloroplast import, the pLHCPII
polyprotein is cleaved into mature LHCPIIs. Due to overlap between
Golgi and thylakoid membrane containing fractions, the possibility can
not be eliminated that processing occurs in a Golgi subcompartment
having the same density as thylakoids but totally distinct from the
other Golgi containing fractions.
Crude cell-free extracts were fractionated into a 3000
Extraction of the 3000
Recovery of in
vivo synthesized pLHCPII in the membrane fraction after
Na
Immunoelectron microscopy provided the first indication that
Euglena has a unique mechanism for chloroplast protein import.
Upon exposure of dark-grown resting Euglena to light,
anti-LHCPII immunogold labeling is first detected over the COS (20, 22,
45), a cytoplasmic osmiophilic structure composed of a central
osmiophilic core with connecting reticulate extensions creating a
compartment containing cytoplasmic material including
ribosomes
(20, 22, 45, 46) . As
chloroplast development proceeds, labeling is detected over the Golgi
apparatus and finally the chloroplast. The chloroplast is the only
organelle labeled in cells that are not actively synthesizing
LHCPII
(20, 21, 22) .
In vivo and
in vitro studies complementing the immunomicroscopy have
further elucidated the novel mechanism of chloroplast protein import
used by Euglena. pLHCPII is synthesized on membrane-bound
polysomes rather than free polysomes, and the presequence 35 amino acid
signal peptide domain is removed during co-translational insertion into
canine microsomal membranes
(18, 19) . At the end of a
10-min pulse, subcellular fractionation localizes pLHCPII to the ER and
Golgi apparatus. Little chloroplast localized pLHCPII is seen at the
end of the pulse, and mature LHCPII is undetectable. During a 20- or
40-min chase, the fraction of ER localized pLHCPII decreases, Golgi
localized pLHCPII increases, and mature LHCPII but not pLHCPII
accumulates in the chloroplast and thylakoid fractions. Diverse
experimental approaches thus provide evidence for Euglena pLHCPII co-translational signal peptide-dependent transport to the
ER and from the ER to the Golgi apparatus prior to chloroplast
localization. This contrasts with the direct post-translational
chloroplast uptake of plant and green algal pLHCPII.
Na
The ER and Golgi apparatus are
sites of endoproteolytic processing of many proproteins and
polyproteins
(23, 24, 25, 26) . Some
Semliki Forest Virus proteins are produced by signal peptidase cleavage
of a polyprotein precursor
(23) . Polyprotein precursors to
neuroendocrine peptide hormones, yeast mating factor, and some
proproteins are processed by trans-Golgi network resident
proteases
(24, 25, 26) . Vacuolar protein
processing is, however, completed by sequence-specific vacuole resident
proteases
(49) .
pLHCPII polyprotein maturation requires
endoproteolytic removal of the presequence and the conserved
decapeptide linker joining individual LHCPIIs within the polyprotein.
Pulse-chase experiments showed that LHCPII was localized only in
chloroplasts and thylakoids, never in the ER and Golgi apparatus.
pLHCPII was predominately ER and Golgi localized. Distinct processing
intermediates, immunoprecipitable proteins with a molecular mass less
than the smallest pLHCPII (116 kDa), were not seen in the Golgi
apparatus during the chase. Unlike maturation of many proproteins and
polyprotein precursors, the pLHCPII signal peptide-like decapeptide
linker is not cleaved by signal peptidase in the ER or by Golgi
resident proteases. As found for vacuole proteins, pLHCPII appears to
be processed to LHCPII upon reaching its final intracellular location,
the chloroplast
(49) .
LHCPII is one of many Euglena chloroplast proteins synthesized in the cytoplasm and transported
to the chloroplast. Amino acid sequences for five Euglena precursors have been deduced from cDNA
sequences
(19, 50, 51, 52, 53) .
The precursor presequences differ but contain common structural
features. Four are about 140 amino acids long, they are enriched in
hyroxylated amino acids, they have a signal peptide domain with a
potential signal peptidase cleavage site 35-50 amino acids from
the N terminus, and they contain at least one additional hydrophobic
domain that could function as a membrane anchor-stop transfer
sequence
(19, 50, 51, 52) . The oxygen
evolving enhancer protein presequence is shorter, 93 amino acids,
contains two hydrophobic domains, but lacks a N-terminal signal
peptide
(53) . Trans-splicing adds a 5` end to all Euglena mRNAs
(54) . The oxygen evolving protein cDNA lacks this
trans-spliced 5` end
(53) indicating it is not full-length. The
smaller size and absence of a N-terminal signal peptide is thus
explained by its being deduced from an incomplete cDNA. The common
structural framework of the Euglena presequences, especially
the signal peptide domain, suggests that all cytoplasmically
synthesized chloroplast proteins are transported as integral membrane
proteins from the ER to the Golgi apparatus and from the Golgi
apparatus to the chloroplast.
In contrast to green algae and higher
plant chloroplasts which are surrounded by two membranes, Euglena chloroplasts are surrounded by three membranes suggesting that
they evolved from a eukaryotic rather than a prokaryotic
endosymbiont
(55) . It has been postulated that the third
chloroplast membrane evolved from the plasma membrane of the original
photosynthetic symbiont, that it evolved from a phagocytic vacuole
membrane, or that it is part of the ER
(55) . Utilization of the
secretory pathway for protein transport from the cytoplasm to the
chloroplast is not surprising when one considers the evolutionary
origin of the third membrane surrounding Euglena chloroplasts.
How integral membrane precursor proteins are transferred from Golgi
transport vesicles through the three membranes surrounding Euglena chloroplasts remains unknown. Transformation of the Euglena proplastid into a chloroplast requires lipids for envelope
expansion and thylakoid formation. The Euglena mutant
W
S]sulfate, organelles separated on sucrose
gradients, and pLHCPII and LHCPII immunoprecipitated and separated on
SDS gels. After a 10-min pulse, the pLHCPII polyprotein was found in
the endoplasmic reticulum (ER) and Golgi apparatus. LHCPII was
undetectable after a 10-min pulse consistent with the 20-min half-life
for pLHCPII processing. When pulse-labeled cells were chased for 20 or
40 min with unlabeled sulfate, the fraction of pLHCPII in the ER
decreased, and the fraction in the Golgi apparatus increased. LHCPII
appeared only in thylakoids and chloroplasts, never in the ER or Golgi
apparatus. Na
CO
extraction, a treatment that
releases soluble but not integral membrane proteins, did not remove
pLHCPII from ER and Golgi membranes. Trypsin digestion of ER and Golgi
membranes produced 4 pLHCPII membrane protected fragments. The
Euglena pLHCPII polyprotein is transported as an integral
membrane protein from the ER to the Golgi apparatus and from the Golgi
apparatus to the chloroplast. Polyprotein processing appears to occur
during or soon after chloroplast import of the membrane-bound
precursor.
(
)
is
co-translational and dependent upon an N-terminal extension, the signal
sequence
(1) . The nascent protein's signal sequence
interacts with signal recognition particle, translation is arrested,
the signal recognition particle-ribosome complex binds an integral ER
membrane protein, elongation resumes, the nascent chain is translocated
into the ER, and the signal sequence is cleaved
(2, 3) .
Integral membrane proteins are co-translationally inserted into the ER
membrane in a linear manner
(1) . A cleaved or uncleaved signal
peptide initiates translocation, and a stop transfer sequence stops
translocation anchoring the protein in the
membrane
(1, 4) . The orientation of proteins with
multiple membrane-spanning domains is determined by the linear
alternating appearance of start transfer membrane anchor sequences and
stop transfer membrane anchor sequences (1, 5, 6). From the ER,
proteins are transported to the Golgi apparatus where they are sorted
for transport to their final destination
(7) .
Cell Growth and Metabolic Labeling
Euglena
gracilis Klebs var. bacillaris Cori maintained in our
laboratory in the dark for many years was used throughout this work.
Conditions for cell growth on low sulfur medium and preparation of dark
grown resting cells were essentially as
described
(28, 29) . After 3 days on resting medium, cell
division had ceased and chloroplast development was initiated by light
exposure
(28) . Growth on low sulfur medium has no effect on
light induced chloroplast development
(29) .
SO
(ICN
Radiochemicals, Irvine, CA) by incubation at 26 °C with shaking in
the light. A chase was initiated by the addition of
K
SO
for a final concentration of 0.1
M. The addition of K
SO
immediately
inhibited any further incorporation of
[
S]sulfate into trichloroacetic acid
precipitable material
(15) .
Cell Fractionation by Isopycnic Sucrose Gradient
Centrifugation
All operations were performed at 0-4
°C. For measurements of marker enzyme activities, 250-ml cells were
harvested by centrifugation at 1000 g for 5 min,
washed with buffer A (25 mM HEPES-KOH, pH 7.4, 1 mM
EDTA, and 400 mM sucrose) and resuspended in 1 ml of buffer
A/g of cells. Cells were broken by grinding for 2.5 min with 2.5 g/ml
acid-washed glass beads (250-300 µm, Sigma). Homogenates were
removed from the settled beads and the beads washed three times with
0.5 ml of buffer A. The pooled homogenates were centrifuged at 150
g for 5 min to remove beads and unbroken cells.
[
S]Sulfate-labeled cells (10 ml) were harvested
at room temperature by centrifugation at 3000
g for 2
min and resuspended in 0.5 ml of buffer A containing the protease
inhibitors (Boehringer Mannheim) antipain (1 µg/ml), chymostatin
(100 µg/ml), pepstatin (10 µg/ml), E64 (1 µg/ml),
phenylmethylsulfonyl fluoride (0.5 mM), aprotinin (1
µg/ml), and leupeptin (1 µg/ml). Cells were broken by grinding
with a glass pestile in a 15-ml plastic conical centrifuge tube
(Nalgene) for 2.5 min with 1.0 g of glass beads. Homogenates were
removed from the settled beads and the beads washed three times with
0.5 ml of buffer A. The pooled homogenates were centrifuged at 150
g for 5 min to remove beads and unbroken cells.
g (26,000 rpm) for 3 h in a Beckman SW 41 rotor and fractionated
from the top into 0.4-ml fractions. Individual fractions were assayed
for marker enzymes or precipitated with 10% trichloroacetic acid, and
the trichloroacetic acid precipitates were solubilized prior to
immunoprecipitation by boiling for 2 min in 100 µl of SDS buffer
(2% SDS, 60 mM Tris-HCl, pH 8.6).
Assay for Membrane Integration by Na
A clarified cell-free
homogenate (1.5 ml) prepared from 10 ml of pulse-labeled cells was
centrifuged at 3,000 CO
Extraction and Trypsin Digestion
g for 10 min to obtain a 3,000
g chlorophyll-free supernatant and a 3,000
g pellet containing all of the chloroplast membranes. The
pellet was resuspended in buffer A while the supernatant was used
directly for Na
CO
extraction and protease
protection assays. Na
CO
and potassium acetate
extraction were performed by adjusting a 0.5-ml aliquot of the 3000
g pellet and supernatant fractions to 0.1 M
Na
CO
pH 11.5 or 0.5 M potassium
acetate, incubating on ice for 30 min, and recovering the extracted
membranes by centrifugation for 1 h at 150,000
g in a
Beckman Ti-70.1 rotor. The 150,000
g membrane pellet
resuspended in buffer A, and the supernatants were precipitated with
10% trichloroacetic acid (w/v). The trichloroacetic acid precipitates
were solubilized prior to immunoprecipitation by boiling for 2 min in
100 µl of SDS buffer.
g supernatant on ice for 30
min with 0.1 mg/ml trypsin (Type XIII, Sigma) in the presence or
absence of 0.5% Triton X-100. An aliquot was incubated in the absence
of trypsin as a control for endogenous proteolysis. Trypsin was added
from a 1 mg/ml stock solution prepared in 50 mM HEPES-KOH, pH
7.5, 100 mM NaCl, 0.2 M sucrose. Proteolysis was
terminated by addition of phenylmethylsulfonyl fluoride for a final
concentration of 10 mM and the membranes recovered by
centrifugation for 1 h at 150,000
g in a Beckman
Ti-70.1 rotor. The membrane pellet was solubilized prior to
immunoprecipitation by boiling for 2 min in 100 µl of SDS buffer.
Immunoprecipitation and SDS-Gel
Electrophoresis
Immunoprecipitation with anti-LHCPII and protein
A-Sepharose (Pharmacia LKB Biotechnology) was performed as described
previously
(15) . Immunoprecipitated proteins were separated on
8-12% linear SDS-polyacrylamide gels
(30) or 12% Tricine
gels
(31) . Gels were impregnated with 1 M sodium
salicylate (32), dried, and exposed to preflashed Kodak X-Omat AR film
at -70 °C. Radioactivity in individual bands was quantified
with a PhosphorImager (Molecular Dynamics).
Analytical Methods
Rotenone-insensitive
NADH-cytochrome c reductase activity was measured at 25 °C
by following the increase in absorbance at 550 nm
(33) . The 1-ml
reaction mixture contained a 100-µl gradient fraction, 50
mM potassium phosphate, pH 7.5, 40 µM cytochrome
c, 1 mM potassium cyanide, and 1.5 µM
rotenone. Glucose-6-phosphatase activity was assayed at 30 °C by
the method of Arion
(34) . The 100-µl reaction mixture
contained a 30-µl gradient fraction, 50 mM sodium
cacodylate buffer, pH 6.5, and 20 mM glucose 6-phosphate
(disodium salt, Sigma). After incubation for 30 min, the reaction was
stopped by addition of 0.9 ml of stop solution composed of 0.42%
ammonium molybdate in 1 N HSO
, 10%
SDS, 10% ascorbic acid (6:2:1, v/v/v), and the amount of inorganic
phosphate released was measured by absorption at 720 nm. Latent IDPase
was determined at 30 °C by the method of Bowles and
Kauss
(35) . The 100-µl reaction mixture contained a
15-µl gradient fraction, 60 mM Tris, pH 7.4, 5 mM
IDP, and 1 mM MgCl
with and without 0.1% (v/v)
Triton X-100. After incubation for 20 min, the reaction was stopped by
addition of 0.9 ml of stop solution, and the amount of inorganic
phosphate released was measured by absorption at 720 nm. Chlorophyll
was determined by measuring autofluorescence
(36) .
Separation of Euglena Subcellular Organelles by
Isopycnic Sucrose Gradient Centrifugation
In vivo studies of the intracellular transport and post-translational
proteolytic processing of pLHCPII required the development of a rapid
method for isolating organelles from small samples of pulse labeled
cells. A number of methods have been developed for preparing
Euglena extracts containing functional intact organelles. The
method of choice is to digest the pellicle for 1 h with trypsin
followed by osmotic disruption (37). The long time (1 h) required for
pellicle weakening precludes using trypsinization to obtain subcellular
organelles for pulse-chase localization studies. An alternative to
osmotic lysis of trypsinized Euglena is to break cells by
grinding with glass beads
(38) . By modifying a previously
published grinding procedure
(38) , a rapid method for preparing
crude cell-free extracts containing 40-60% intact chloroplasts
was developed. Within 5-7 min of harvest, crude cell-free
extracts could be prepared from pulse-labeled cells and loaded onto
sucrose gradients for isopycnic separation of subcellular organelles.
Figure 1:
Localization of
Euglena organelles on isopycnic sucrose density gradients. A
crude cell homogenate (1.5 ml) prepared from dark-grown Euglena exposed to light for 24 h was loaded onto a 12-ml gradient
consisting of a 2-ml 20% (w/w) sucrose step on top of a 25-50%
(w/w) linear sucrose gradient formed over a 0.5-ml cushion of 55% (w/w)
sucrose. Gradients were centrifuged at 100,000 g (26,000 rpm) for 3 h in a Beckman SW 41 rotor and fractionated
from the top into 0.4-ml fractions. Organelles were identified by
measuring the following activities: chlorophyll, a marker for
thylakoids and intact chloroplasts; SSU, a marker for intact
chloroplasts and soluble fraction (released from broken chloroplasts);
rotenone-insensitive NADH cytochrome c reductase (NADH cyt
c red), an ER and mitochondrial marker; glucose-6-phosphatase
(Glc-6-Pase), an ER marker; latent IDPase, a Golgi
marker.
Latent IDPase, a Golgi marker enzyme
(39) ,
formed two peaks between fractions 10-18 (Fig. 1). The
second IDPase peak was always less dense and resolved from thylakoid
membranes. There was no overlap between Golgi membrane peaks and intact
chloroplasts. Golgi membranes from Euglena(39) and
other algae
(40, 41) are often resolved into multiple
fractions of different density. It is not known whether these different
density fractions represent functionally distinct Golgi compartments.
(
)
a mitochondrial inner membrane
protein, was found in fractions 20-23 identifying them as
containing intact mitochondria. A minor peak of rotenone-insensitive
NADH cytochrome c reductase was found in fractions 8-11
between the ER and Golgi membranes (Fig. 1).
Glucose-6-phosphatase was not present in these fractions eliminating
the possibility that they contain ER membranes. Succinate dehydrogenase
was not found in fractions 8-11.
The presence of NADH
cytochrome c reductase and absence of succinate dehydrogenase
from fractions 8-11 suggests that mitochondrial outer membranes
band in this region of the gradient.
Intracellular Transport and Subcellular Site of pLHCPII
Polyprotein Processing
Euglena pLHCPIIs are a group of
extremely large polyproteins having molecular masses of 110, 122, 161,
and 207 kDa
(15) that are converted to LHCPIIs with a 20-min
half-life
(15) . When cells exposed to light for 24 h were pulse
labeled for 10 min with [S]sulfate, the pLHCPIIs
are the only proteins immunoprecipitated (Fig. 2A). The
26- and 27-kDa mature LHCPIIs
(15) are undetectable. After a
20-min chase with unlabeled sulfate, pLHCPIIs and mature LHCPIIs are
found in the immunoprecipitate (Fig. 2B) while after a
40-min chase, mature LHCPIIs are the predominant proteins
immunoprecipitated (Fig. 2C). In order to determine the
intracellular route of pLHCPII transport from the cytoplasm to the
chloroplast and the intracellular site of polyprotein processing,
cell-free extracts were prepared from
[
S]sulfate-labeled cells, organelles were
separated by isopycnic density gradient centrifugation, and pLHCPII and
LHCPII were immunoprecipitated from each fraction and separated by SDS
gel electrophoresis.
Figure 2:
Intracellular localization of pLHCPII and
LHCPII. Dark-grown Euglena exposed to light for 24 h were
pulse labeled for 10 min with [S]sulfate. A
chase was initiated by addition of unlabeled sulfate for a final
concentration of 0.1 M. Cell-free extracts were prepared at
the end of the 10-min pulse (A), 20 (B), or 40 min
© after the start of the chase. Organelles were separated by
isopycnic centrifugation on 12-ml gradients consisting of a 2 ml 20%
(w/w) sucrose step on top of a 25-50% (w/w) linear sucrose
gradient formed over a 0.5-ml cushion of 55% (w/w) sucrose. After
centrifugation at 100,000
g (26,000 rpm) for 3 h in a
Beckman SW 41 rotor, gradients were fractionated from the top into
0.4-ml fractions. Each fraction was precipitated with 10%
trichloroacetic acid, resuspended by boiling in 2% SDS, and an aliquot
was immunoprecipitated with antibody to Euglena LHCPII. An
aliquot of the crude cell-free extract loaded onto the gradient was
also immunoprecipitated. The immunoprecipitates were analyzed on
8-12% linear gradient Tris-glycine SDS-polyacrylamide gels and
the immunoprecipitated proteins visualized by fluorography. The regions
of the fluorographs containing pLHCPII and LHCPII are
presented.
The subcellular localization of LHCPII was
distinctly different from pLHCPII. (Fig. 2). pLHCPIIs were found
predominately in the ER, fractions 3-6, and Golgi apparatus,
fractions 10-18, while LHCPII was found predominately in
thylakoids, fractions 15-18, and chloroplasts, fractions
19-22 (Fig. 2). All four pLHCPIIs were found in each
pLHCPII containing fraction. ER localized pLHCPIIs were degraded while
pLHCPIIs in other regions of the gradient were relatively undegraded
(Fig. 2). Organelle breakage occurred during cell disruption
releasing organelle proteases into the soluble cytoplasmic fraction.
Degradation of ER localized pLHCPII probably results from incomplete
separation of the ER fraction from the protease containing soluble
fraction remaining at the top of the gradient. The inclusion of
protease inhibitors in the isolation and gradient buffers did not fully
inhibit pLHCPII degradation.
S-labeled protein did not change during the chase, but the
extent of cell breakage and thus the fraction of total cellular
S-labeled protein in the homogenate varied from sample to
sample. To allow direct comparisons between gradients loaded with
differing amounts of
S-labeled protein, the amount of
pLHCPII and LHCPII in each fraction is plotted in Fig. 3as the
percent of total immunopreciptate (pLHCPII and LHCPII) recovered from
the gradient.
Figure 3:
Quantitative analysis of pLHCPII and
LHCPII intracellular localization. The SDS gels presented in Fig. 2
from a 10-min pulse (A), a 20-min (B) or 40-min chase
were scanned with a PhosphorImager. pLHCPII levels are defined as the
sum of the image intensity for the 207-, 161-, 122-, and 106-kDa
pLHCPIIs and LHCPII levels are defined as the sum of the image
intensity for the 26- and 27-kDa LHCPIIs. To allow direct comparisons
between gradients loaded with differing amounts of
S-labeled protein, the amount of pLHCPII and LHCPII in
each fraction is plotted as a percent of the total immunoprecipitate
(pLHCPII and LHCPII) recovered from the
gradient.
After a 10-min pulse, a pLHCPII peak was found in
fractions 3-6, ER membranes, a shoulder in fractions 8-12,
low density Golgi membranes, and a second peak in fractions
13-18, high density Golgi membranes (Fig. 2A and
3A). The distribution of pLHCPII between the low and high
density Golgi fractions varied from experiment to experiment while the
total fraction of Golgi localized pLHCPII remained relatively constant.
The major pLHCPII peaks were less dense and resolved from the two
chlorophyll peaks used as internal standards to locate thylakoids and
chloroplasts (Fig. 3A). Extensive degradation of ER
localized pLHCPII (Fig. 2) results in a large fraction of
immunoprecipitable radioactivity having molecular weights differing
from that of the four pLHCPIIs. These degraded pLHCPIIs are not
resolved as distinct polypeptides and could not be quantitated by the
software employed resulting in a significant underestimate of ER
localized pLHCPII. The reported amount (Fig. 3) of ER localized
pLHCPII is thus a minimum estimate. The absence of degradation
(smearing) in other gradient regions allows accurate determinations of
Golgi, thylakoid, and chloroplast localized pLHCPII (Fig. 3).
The Euglena pLHCPII Polyprotein Is Transported to the
Chloroplast as an Integral Membrane Protein
Euglena pLHCPII synthesized in vitro is co-translationally
inserted into canine microsomes, processed by signal peptidase, and
anchored within the membrane remaining protease accessible on the
cytoplasmic membrane face
(19) . Alkaline NaCO
extraction converts membrane vesicles into open membrane sheets
releasing luminal and peripheral proteins
(43) .
Na
CO
extraction and protease protection assays
have been used to determine whether in vivo synthesized
pLHCPII is an integral ER and Golgi protein with protease accessible
cytoplasmic domains as suggested by the in vitro processing
studies.
g pellet containing 87% of the chlorophyll, 14% of the
IDPase, and 40% of the glucose-6-phosphatase activity and a 3000
g supernatant containing only 3% of the chlorophyll,
90% of the IDPase, and 60% of the glucose-6-phosphatase activity. Based
on the marker enzyme distribution, the 3000
g pellet
contains thylakoids, chloroplasts, and ER membranes while the 3000
g supernatant is a chloroplast membrane-free ER and
Golgi fraction. Since gradient fractionation localized pLHCPII to the
ER and Golgi apparatus with little in thylakoids and chloroplasts
(Fig. 3), the 3000
g pellet was used to study
the topology of ER localized pLHCPII. Membranes often aggregate during
pelleting sequestering proteins within the aggregate making them
inaccessible to exogenous protease
(44) . Possible aggregation
artifacts were avoided by using the 3000
g supernatant
as an ER-Golgi membrane fraction.
g pellet and supernatant with 0.5 M potassium acetate
or 0.1 M Na
CO
did not remove pLHCPII
from ER and Golgi membranes (Fig. 4A). All four pLHCPIIs
were recovered in the acetate and Na
CO
extracted membrane pellet, and pLHCPIIs were barely detectable in
the membrane-free supernatant (Fig. 4A). PhosphorImager
analysis of the gels indicated that 95% of the two larger pLHCPIIs and
87% of the two smaller pLHCPIIs remained membrane associated after the
3000
g pellet was extracted with potassium acetate.
Extraction of the 3000
g supernatant with potassium
acetate resulted in 75% of the two larger and 89% of the two smaller
pLHCPIIs remaining membrane-associated. Na
CO
extraction of the 3000
g pellet resulted in 95%
of the two larger and only 64% of the two smaller pLHCPIIs remaining
membrane associated while 78% of the two larger and only 56% of the two
smaller pLHCPIIs were recovered in the membrane pellet after extracting
the 3000
g supernatant with
Na
CO
. The recovery of pLHCPII with
Na
CO
extracted ER and Golgi membranes
identifies pLHCPII as an integral membrane protein.
Figure 4:
Potassium acetate and
NaCO
extractability of organelle-associated
pLHCPII, LHCPII, and SSU. Euglena exposed to light for 24 h
was pulse labeled for 10 min with [
S]sulfate
(A) or for 30 min with [
S]sulfate
(B). A cell-free extract was centrifuged at 3000
g for 10 min producing a chloroplast and ER membrane containing 3000
g pellet and a chlorophyll-free ER and Golgi membrane
containing 3000
g supernatant. A, the
resuspended 3000
g pellet (lanes 1-5)
and supernatant (lanes 6-10) were adjusted to 0.5
M potassium acetate (Ac) or 0.1 M
Na
CO
, pH 11.5 (CO
), the
extracted membranes recovered by centrifugation, and pLHCPII in the
membrane pellet (P) and supernatant (S) was
immunoprecipitated. B, the resuspended 3000
g pellet (lane 1) was adjusted to 0.5 M potassium
acetate (Ac, lanes 2 and 3) or 0.1
M Na
CO
, pH 11.5 (CO
lanes 4 and 5), the extracted membranes
recovered by centrifugation and LHCPII and SSU in the membrane pellet
(P) and supernatant (S) were immunoprecipitated.
Immunoprecipitates were analyzed on 8-12% (pLHCPII and LHCPII) or
8-16% (SSU) linear gradient Tris-glycine SDS-polyacrylamide gels.
The regions of the fluorographs containing pLHCPII (A), LHCPII
(B), and SSU (B) are
presented.
To confirm that
NaCO
extraction can distinguish soluble from
integral membrane proteins, the extractability of the small subunit of
ribulose bisphosphate carboxylase (SSU), a soluble chloroplast protein,
and LHCPII, an integral thylakoid membrane protein was determined
(Fig. 4B). A lack of characterized ER and Golgi
localized Euglena proteins necessitated using chloroplast
proteins for these experiments. SSU was recovered in the membrane
pellet when chloroplasts in the 3000
g pellet were
extracted with potassium acetate and SSU was recovered in the
membrane-free supernatant after extracting chloroplasts with 0.1
M Na
CO
(Fig. 4B).
LHCPII on the other hand was recovered in the membrane pellet when
chloroplasts were extracted with either potassium acetate or
Na
CO
(Fig. 4B). The removal of
SSU but not LHCPII from chloroplasts by Na
CO
extraction demonstrates that under the conditions used in these
experiments, Na
CO
extractability distinguishes
soluble proteins from integral membrane proteins.
CO
extraction demonstrated that pLHCPII is
anchored in the ER and Golgi membrane. Protease protection assays were
performed in order to obtain information about the topology of pLHCPII
within the membrane. pLHCPIIs were not degraded by endogenous proteases
during incubation of the 3000
g supernatant on ice
(Fig. 5, A and B). Trypsin digested all four
pLHCPIIs. Four polypeptides, 24, 22, 15, and 10 kDa were recovered with
trypsin-digested membranes only in the absence of detergent
demonstrating that they are membrane protected digestion products
(Fig. 5, A and B). Immunoprecipitable peptides
were undetectable in the membrane-free supernatant.
Analysis of the digestion products on Tricine gels did not detect
additional low molecular weight protease protected fragments
(Fig. 5B). Trypsin digestion of the 3000
g pellet produced the same protected fragments as obtained from
digestion of the 3000
g supernatant.
Two
of the protected fragments, 24 and 22 kDa, are similar in size to
thylakoid-protected LHCPII trypsin digestion products.
Taken together, the protease protection and
Na
CO
extraction experiments indicate that
multiple membrane-spanning regions anchor pLHCPII in the ER and Golgi
membrane.
Figure 5:
Trypsin digestion of ER and Golgi
localized pLHCPII. Euglena exposed to light for 24 h was pulse
labeled for 10 min with [S]sulfate. A
chlorophyll-free ER and Golgi membrane containing 3000
g supernatant (lane 1) was digested with trypsin (0.1
mg/ml) in the absence or presence of 0.5% Triton X-100. Proteolysis was
terminated by addition of phenylmethylsulfonyl fluoride for a final
concentration of 10 mM and the membranes recovered by
centrifugation. pLHCPII-protected fragments were immunoprecipitated and
separated on 8-12% linear gradient Tris-glycine
SDS-polyacrylamide gels (A) or 12% Tricine SDS gels
(B) and the immunoprecipitated peptides visualized by
fluorography.
CO
did not remove pLHCPII from Euglena ER and Golgi membranes demonstrating it is an integral membrane
protein. Trypsin digestion produced four protease-protected pLHCPII
fragments. The individual LHCPIIs within the polyprotein are highly
homologous
(16, 17) . Trypsin cleavage sites are found at
similar positions within each mature LHCPII unit of the polyprotein.
Each protease-protected fragment may be derived from more than one
LHCPII unit. The two largest protease-protected fragments (22 and 24
kDa) are similar in size to the protected fragments produced by trypsin
digestion of Euglena thylakoids. Higher plant LHCPIIs are
inserted into thylakoids post-translationally and the three
membrane-spanning domains orient the protein with the N terminus in the
stroma and C terminus in the
lumen
(12, 13, 14, 47) . Euglena and higher plant LHCPIIs are not inserted into microsomal
membranes or bacterial inner
membranes
(13, 19, 48) . A chimeric plant LHCPII
containing a bacterial signal peptide at its N terminus is, however,
inserted into the bacterial inner membrane and anchored within the
membrane by hydrophobic sequences within LHCPII
(48) . Signal
peptide initiated linear insertion of the chimera into the membrane
appears to enable one or all of the hydrophobic LHCPII domains to be
inserted within the membrane. Euglena pLHCPII is
co-translationally inserted into canine microsomal membranes, and
signal peptidase removes the first 35 N-terminal amino acids of the
presequence
(19) demonstrating that it is a naturally occurring
signal peptide-LHCPII chimera. Based on the similarity in size between
the two largest pLHCPII ER-Golgi-protected fragments and the LHCPII
thylakoid-protected fragments, it is tempting to speculate that some if
not all LHCPII units within the pLHCPII polyprotein are anchored within
the ER and Golgi membrane in the same orientation as in thylakoids,
spanning the membrane three times with the N terminus in the cytoplasm
and the C terminus in the lumen.
BUL lacks most if not all of the chloroplast genome but
contains a proplastid remnant that upon light exposure undergoes
limited development
(56) suggesting a cytoplasmic origin for
chloroplast lipid. Electron microscopy found membrane aggregates in the
form of membrane whorls and osmiophilic bodies in the cytoplasm of dark
grown Euglena(56, 57, 58) . Shortly
after initiation of proplastid development, the membrane whorls are
seen extending from the interior of the prolammellar body, the
osmiophilic bodies are fused to the proplastid envelop, and the
osmiophilic membrane aggregates are dispersed in the
stroma
(57, 58) . Since protease protection assays
suggest that pLHCPII is integrated into the Golgi membrane in the same
orientation as found within the thylakoid, it is tempting to speculate
that Euglena thylakoid assembly is initiated within the ER and
Golgi apparatus. Interaction between precursor proteins or their
presequences within the Golgi transport vesicles could cause vesicle
aggregation producing the membrane whorls observed by electron
microcopy. Internalization of these precursor containing transport
vesicles through fusion with the proplastid envelop membrane, release
of stromal proteins from the internalized vesicles by presequence
cleavage, migration of thylakoid membrane proteins within the lipid
bilayer, and integration of chloroplast synthesized thylakoid proteins
into the developing thylakoid would complete thylakoid assembly
initiated within the ER and Golgi apparatus. Although highly
speculative, this model is consistent with ultrastructural and
biochemical studies of Euglena chloroplast biogenesis
providing a mechanism for chloroplast import of membrane bound
precursors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.