Journal of Experimental Botany, Vol. 58, No. 6, pp. 1365–1379, 2007
doi:10.1093/jxb/erl303 Advance Access publication 23 February, 2007
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Intracellular sorting of the tail-anchored protein cytochrome
b5 in plants: a comparative study using different isoforms
from rabbit and Arabidopsis
Caterina Maggio1, Alessandra Barbante1, Flavia Ferro1, Lorenzo Frigerio2 and Emanuela Pedrazzini1,*
1
2
CNR Istituto di Biologia e Biotecnologia Agraria, via Bassini 15, Milano, Italy
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 21 September 2006; Revised 14 December 2006; Accepted 18 December 2006
Abstract
Tail-anchored (TA) proteins are bound to membranes
by a hydrophobic sequence located very close to the
C-terminus, followed by a short luminal polar region.
Their active domains are exposed to the cytosol. TA
proteins are synthesized on free cytosolic ribosomes
and are found on the surface of every subcellular
compartment, where they play various roles. The basic
mechanisms of sorting and targeting of TA proteins to
the correct membrane are poorly characterized. In
mammalian cells, the net charge of the luminal region
determines the sorting to the correct target membrane,
a positive charge leading to mitochondria and negative
or null charge to the endoplasmic reticulum (ER). Here
sorting signals of TA proteins were studied in plant
cells and compared with those of mammalian proteins,
using in vitro translation–translocation and in vivo
expression in tobacco protoplasts or leaves. It is shown
that rabbit cytochrome b5 (cyt b5) with a negative
charge is faithfully sorted to the plant ER, whereas
a change to a positive charge leads to chloroplast
targeting (instead of to mitochondria as observed in
mammalian cells). The subcellular location of two cyt
b5 isoforms from Arabidopsis thaliana (At1g26340
and At5g48810, both with positive net charge) was
then determined. At5g48810 is targeted to the ER, and
At1g26340 to the chloroplast envelope. The results
show that the plant ER, unlike the mammalian ER, can
accommodate cytochromes with opposite C-terminal
net charge, and plant cells have a specific and as
yet uncharacterized mechanism to sort TA proteins
with the same positive C-terminal charge to different
membranes.
Key words: Chloroplast envelope, cytochrome b5, membrane
proteins, protein sorting, tail-anchored proteins.
Introduction
Tail-anchored (TA) proteins are a class of polypeptides
that are inserted into membranes by a C-terminally located
hydrophobic sequence and have their functional domains
exposed to the cytosolic face of membranes. TA proteins
are synthesized on free ribosomes in the cytosol and do
not interact with canonical translocation systems because
their hydrophobic domain emerges from the ribosome
only when the translation is completed. Proteins of this
class are sorted to their target membranes by posttranslational mechanisms that are still poorly characterized
(Borgese et al., 2003).
In all eukaryotes, TA proteins carry out a variety of
essential functions, such as the regulation of vesicular traffic
(Lauber et al., 1997; Geelen et al., 2002), electron transfer
reactions (Borgese et al., 1993a; Mullen et al., 2000), and
regulation of apoptosis (Cory and Adams, 2002).
TA proteins resident in compartments and vesicles of
the secretory pathway are first inserted into the endoplasmic reticulum (ER) membrane, then travel along
the endomembrane system according to their physicochemical features, until they reach their final destination
(Pedrazzini et al., 1996). The available evidence indicates
that TA proteins destined to membranes of semi-autonomous
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: CaMV, cauliflower mosaic virus; COE, chloroplast outer envelope; cyt b5, cytochrome b5; Endo H, endoglycosidase H; ER, endoplasmic
reticulum; GFP, green fluorescent protein; mAb, monoclonal antibody; MOM, mitochondrial outer membrane; PCR, polymerase chain reaction; TA, tailanchored; YFP, yellow fluorescent protein.
ª 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1366 Maggio et al.
organelles or peroxisomes [mitochondrial outer membrane (MOM), peroxisomal membrane, chloroplast outer
envelope (COE)] reach these membranes directly from the
cytosol, without passing through the ER (Borgese et al.,
2001). Therefore, targeting of TA proteins must involve
mechanism(s) that discriminate between the ER membrane and membranes of organelles that are not part of the
secretory system. Recently, some progress has been made
to solve this problem in animal cells; however, only very
limited information is available on the mechanisms of TA
protein sorting in plants.
A useful model to study the sorting and targeting of TA
proteins is the electron carrier cytochrome b5 (cyt b5), one
of the best characterized members of this class (Borgese
et al., 1993b; D’Arrigo et al., 1993). The cyt b5 family
includes 15–23 kDa polypeptides with an N-terminal, globular, cytosolic haem-binding domain, a short connecting region, and a hydrophobic transmembrane domain
followed by a few polar luminal residues at the extreme
C-terminus. Using two differently located forms of mammalian (rabbit) cyt b5, localized on the ER or the MOM, it
has been demonstrated that the short, C-terminal polar
region downstream of the transmembrane domain plays a
crucial role in discriminating between these two destinations (Mitoma and Ito, 1992; Kuroda et al., 1998; Borgese
et al., 2001). This region, which is translocated across the
phospholipid bilayer, carries a net negative charge in ERtargeted cyt b5 but not in the MOM form; the alteration to
a positive charge results in mistargeting of the ER form to
the MOM (Borgese et al., 2001).
Sequence analysis of cyt b5 isoforms from different
organisms revealed that the net negative charge of the Cterminal polar region, characteristic of the mammalian ER
isoform, is not present in cyt b5s of most other organisms.
Cyt b5 of invertebrates, plants, and fungi generally carries
a neutral or net positive charge in the C-terminal polar
domain (Borgese et al., 2001). This suggests that in plants
and other non-mammalian organisms, the distinction between ER and MOM operates through mechanisms other
than C-terminal charge-based sorting.
With the aim of acquiring information on the targeting
of TA proteins in plants, in the present work the targeting
of two rabbit cyt b5 forms was studied in plant cells. The
results indicate that the mammalian ER and MOM cyt b5
isoforms retain a distinct localization in plant cells. The
ER form is correctly inserted into the ER membrane, but
the MOM form is localized on the COE.
The intracellular distributions of two Arabidopsis cyt b5
isoforms coded by the genes At1g26340 and At5g48810,
both with a positive C-terminus, were also analysed. The
fluorescent reporter green fluorescent protein (GFP) or
yellow fluorescent protein (YFP) was fused at the Nterminus of both to facilitate the studies in vivo and
in vitro. The results show that YFP–At5g48810 is located
on the ER membrane, while YFP–At1g26340 is targeted
to the COE. Both fusions are able to translocate their
C-terminus in plant microsomes during in vitro posttranslational incubation. Moreover, it was found that when
chloroplasts are absent, YFP–At1g26340 is redirected to
the MOM, indicating a potential promiscuity of targeting
and a competition between COE and MOM to capture this
cyt b5 isoform.
Materials and methods
Plasmid constructions
DNA manipulations were carried out by standard techniques
(Ausubel et al., 1987; Sambrook et al., 1989). The absence of
errors in fragments generated by polymerase chain reaction (PCR)
or in synthetic oligonucleotide cassettes was verified by sequencing.
The rabbit cyt b5 forms b5-Nglyc and b5-RRNglyc were derived
from the plasmid pCB6, described in Borgese et al. (2001). To
express this cDNA as well as the other b5 constructs transiently, in
plant cells, the b5 insert was placed under the control of the 35S
cauliflower mosaic virus (CaMV) promoter by subcloning it into
pDHA vector (Tabe et al., 1994). The coding sequences of b5Nglyc and b5-RRNglyc were excised from the pCB6 vector by
digestion with BglII and BamHI, and subcloned into pDHA linearized with BamHI (BglII and BamHI are compatible ends). The
resulting constructs are termed pDHA-b5-Nglyc and pDHA-b5RRNglyc, and are used for tobacco leaf protoplast transformation.
To identify the A. thaliana cyt b5 polypeptides, the mammalian
ER cyt b5 amino acid sequence was blasted against the entire
Arabidopsis proteome of the TAIR site (Agi proteins), using the
BLASTP function. Five putative cyt b5s were found and their cDNA
coding sequences were determined. In this work, two of these
cDNAs were used: At1g26340 and At5g48810, and their cDNA
sequences were kindly provided by TAIR cDNA stock order
service. The At1g26340 and At5g48810 cDNAs were provided
inserted into the pUniT3-D/V5-His-TOPO vector and the pUni51
vector, respectively. To subclone these sequences into the expression pDHA vector, At1g26340 and At5g48810 cDNA were
amplified by PCR standard reaction using the following primers:
At1g26340 forward primer, 5#-GCTCTAGATGTGGATCCGGTGGTATGCCGACACTCACAAAGC-3#; reverse primer, 5#-ACATGCATGCGAGCTCCTCGAGTTAAGTCTTGCGAGAGAACAAG-3#;
At5g48810 forward primer, 5#-GCTCTAGATGTGGATCCGGTGGTATGGGCGGAGACGGAAAA-3#; reverse primer, 5#-ACATGCATGCGAGCTCCTCGAGTCAAGAAGAAGGAGCCTTGGT-3#.
The PCR products were digested with XbaI (at the 5# end) and
SphI (at the 3# end) and subcloned into pDHA digested with XbaI
and SphI. The resulting constructs were termed pDHA-At1g and
pDHA-At5g.
To fuse the N-terminus of Arabidopsis cyt b5 sequences in-frame
with the GFP or YFP coding sequence (mGFP5 or EYFP, Clontech,
Palo Alto, CA, USA), a three-way ligation was performed between
At1g26340 or At5g48810 digested with BamHI/SphI, GFP or YFP
cDNA digested with XbaI/BamHI, and pDHA digested with
XbaI/SphI. The resulting constructs were termed pDHA-GFP::At1g,
pDHA-GFP::At5g, pDHA-YFP::At1g, and pDHA-YFP::At5g.
The EcoRI fragment excised from the pDHA-YFP::At1g or
pDHA-YFP::At5g construct, which contain the 35S CaMV promoter,
the coding sequence for cyt b5 forms, and the 35S terminator, was
subcloned into EcoRI-linearized pGreenII0179 (http://www.pgreen.
ac.uk, John Innes Centre, Norwich, UK) which has the hygromycin
resistance gene. These constructs, called pGreen-YFP::At1g and
pGreen-YFP::At5g, were used to transform Agrobacterium
Sorting of tail-anchored proteins in plants 1367
tumefaciens strain C58 by the freeze–thaw method (An et al.,
1988). A HindIII–EcoRI fragment containing the OEP7:GFP
marker (Lee et al., 2001) expression cassette was cloned into the
same sites of pGreenII0029 for agroinfiltration. This and all other
constructs used for agroinfiltration were also transformed into A.
tumefaciens C58.
To tag the 3# end of At1g26340 and At5g48810 with the OP3
sequence, AgeI/XhoI restriction sites were introduced and the stop
codon was first eliminated by PCR amplification using the following reverse primers (the forward primers were the same as described
before) and Turbo Pfu polymerase (Stratagene): At1gDStop reverse
primer, 5#-AGCTCCTCGAGCTGCAGACCGGTAGAAGAAGGAGC-3#; At5gDStop reverse primer, 5#-AGCGCACTCGAGCTGCAGACCGGTAGTCTTGCGAGAG-3#.
The blunt PCR products were subcloned into the EcoRV site of
pBlueScript KS+. The resulting constructs were termed pBSAt1gDStop and pBs-At5gDStop. pBS-At1gDStop and pBS-At5gDStop were digested with AgeI/XhoI, and a synthetic sequence
obtained by pairing the following two phosphorylated complementary oligonucleotides were introduced: upper synthetic oligonucleotide
(OP3+), 5#PHO-CCGGTATGAACGGAACAGAAGGACCAAACTTCTACGTACCATTCAGCAACAAAACAGTAGACTGAC-3#;
lower synthetic oligonucleotide (OP3–), 5#PHO-TCGAGTCAGTCTACTGTTTTGTTGCTGAATGGTACGTAGAAGTTTGGTCCTTCTGTTCCGTTCATA-3#.
The resulting constructs, termed pBS-At1g-Nglyc and pBS-At5gNglyc, were used for in vitro transcription from the SP6 promoter.
Antibodies
Polyclonal antibodies against GFP were from Molecular Probes
(Eugene, OR, USA). Monoclonal antibody (mAb) against the Nterminal peptide of bovine opsin was kindly provided by Dr Paul
Hargrave (Adamus et al., 1991). Polyclonal anti-BiP antiserum was
raised against a recombinant fusion between maltose-binding protein
and amino acids 551–667 of tobacco BiP (Pedrazzini et al., 1997).
In vitro transcription and translation
b5-Nglyc and b5-RRNglyc in pGEM4 and At1g-Nglyc, and At5gNglyc in pBSKS were transcribed from the SP6 promoter. In vitro
transcription of linearized plasmids was performed as previously
described (Ceriotti et al., 1991).
The resulting synthetic RNAs were translated for 1 h at 32 C in
10 ll or 20 ll of reticulocyte lysate (Promega, Madison, WI, USA)
in the presence of [35S]methionine (translation grade, Amersham
Bioscences) as previously described (Ceriotti et al., 1991). In some
cases, microsomes were added post-translationally: translation was
blocked with RNase and equal amounts of the translation products
(calculated by scanning densitometry) were incubated for 1 h at
25 C in the presence of 2 ll of microsomes from Phaseolus
vulgaris (PhasMR; Lupattelli et al., 1997). Translation–translocation
products were analysed by 15% acrylamide SDS–PAGE and
fluorography.
Transient transformation of protoplasts
Protoplasts were prepared from axenic leaves (4–7 cm long) of
tobacco cv. Petit Havana SR1 and subjected to polyethylene glycolmediated transfection as described by Pedrazzini et al. (1997). A
40 lg aliquot of recombinant pDHA plasmids or pDHA without
inserts (as a negative control for transfection) were used to
transform protoplasts at a concentration of 106 cells ml 1. After
transfection, protoplasts were allowed to recover overnight in the
dark at 25 C in K3 medium (Pedrazzini et al., 1997) at a concentration of 106 cells ml 1 before pulse–chase experiments, fixation,
or protoplast lysis were performed.
Treatment with endoglycosidase H (Endo H)
Transfected protoplasts expressing the selected construct were lysed
24 h after transfection with 2 vols of ice-cold lysis buffer (150 mM
TRIS-HCl, 150 mM NaCl, 1.5 mM EDTA, and 1.5% Triton X-100,
pH 7.5) supplemented with complete protease inhibitor cocktail
(Roche). After clarification by centrifugation at 500 g for 5 min, the
Triton-soluble proteins corresponding to 300 000 protoplasts were
subjected to digestion with 2000 U of Endo H from New England
Biolabs (Beverly, MA, USA), using the reagents and the protocol
supplied by the manufacturer. After 1 h of incubation, the samples were supplemented with Laemmli denaturation buffer and
analysed by SDS-PAGE and protein blotting or SDS-PAGE and
fluorography.
In vivo labelling of protoplasts and analysis of proteins
Radioactive labelling of protoplasts was performed using Pro-Mix
(a mixture of [35S]methionine and [35S]cysteine; Amersham Biosciences, Little Chalfont, UK) as described by Pedrazzini et al.
(1997).
Protoplast homogenization was performed by adding to frozen
samples 2 vols of ice-cold homogenization buffer (150 mM TRISHCl, 150 mM NaCl, 1.5 mM EDTA, and 1.5% Triton X-100, pH 7.5)
supplemented with complete protease inhibitor cocktail (Roche).
Immunoselection with anti-GFP antibodies was performed as described previously (D’Amico et al., 1992) using protein A–Sepharose
(Amersham Biosciences). Radioactive samples were analysed by
SDS-PAGE on 15% acrylamide gels. Rainbow 14C-methylated
proteins (Amersham Biosciences) were used as molecular weight
markers. Radioactive polypeptides were revealed by fluorography.
Gels usually were treated with 2,5-diphenyloxazole dissolved in
dimethylsulphoxide (DMSO) and dried. Alternatively, gels were dried
without treatment and exposed using the intensifying screen
BioMax Transcreen LE (Kodak, Rochester, NY, USA). Measurement of the relative intensities of the bands in the fluorographs was
determined by microdensitometry. Care was taken to use film
exposures that were in the linear range of film darkening.
Subcellular fractionation
At 24 h after transfection 23106 protoplasts were pelleted by
addition of 4 vols of W5 medium (Pedrazzini et al., 1997) and
centrifuged at 50 g for 10 min. The protoplasts were resuspended in
0.8 ml of Differential C buffer (DiffC; 0.5 M sorbitol, 50 mM
HEPES, 1/25 Complete Boehringer, pH 7.0). Protoplast lysis was
achieved using a 2.5 ml syringe fitted with a 10 lm nylon mesh
held in place over the open end of the barrel with a rubber O-ring.
The protoplasts were slowly drawn into the syringe and then slowly
ejected through the mesh. The suspension was examined under a
light microscope after each passage to determine the minimum
number of passages necessary to achieve complete breakage. Usually, two passages through the 10 lm mesh or three passages through
the 20 lm mesh, respectively, were sufficient to break most of the
protoplasts. Cell fractions were recovered by centrifuging the lysate
at 500 g for 2 min at 4 C, using a swinging bucket rotor. The
pellet was resuspended into 0.8 ml of DiffC and called P1. The
supernatant was collected and centrifuged again at 100 000 g for
30 min, 4 C, using an SW55 rotor (Beckman) to recover the
microsomal fraction. The pellet was resuspended into 80 ll of
DiffC. This pellet comprised the microsomal and mitochondrial fractions (P2), and the supernatant comprised the cytosolic and vacuolar
fractions (PNS2). In each fraction, the total protein concentration
was determined with Sigma Bradford Reagent. The subcellular
fractions were aliquoted into small volumes and stored at 70 C.
For SDS-PAGE and protein blotting analysis, equal amounts of
total protein from each fraction were loaded on the gels.
1368 Maggio et al.
Immunofluorescence, agroinfiltration, and confocal
microscopy
At 24 h after transfection, 106 protoplasts expressing b5-Nglyc, b5RRNglyc, or control protoplasts were washed with 4 vols of W5
medium, pelletted for 10 min at 600 rpm, and resuspended in MaCa
buffer [0.5 mM mannitol, 20 mM CaCl2, and 0.1% (w/v) MES,
pH 5.7] containing 4% (w/v) paraformaldehyde for 2 h at room
temperature. After fixation, cells were washed with 4 vols of W5
medium and permeabilized with TSW buffer (10 mM TRIS–HCl
pH 7.4, 0.4 M NaCl, 0.25% gelatin, 0.02% SDS, 0.3% Triton X100) for 30 min. Then 33105 cells were incubated with the
appropriate primary antibody diluted in TSW for 1 h at room
temperature. Primary antibodies were: the mAb anti-OP3 (Adamus
et al., 1991; dilution 1:300) or the polyclonal anti-BiP antiserum
(Pedrazzini et al., 1997; dilution 1:1000) in combination with antiOP3 mAb (for the double staining). After three washes in TSW,
cells were incubated for 1 h at room temperature with Alexa Fluor
488 goat anti-mouse highly cross-adsorbed secondary antibody
alone or Alexa488 goat anti-mouse plus the Alexa Fluor 568 goat
anti-rabbit highly cross-adsorbed secondary antibody, both at a
dilution of 1:1000 (Invitrogen-Molecular Probes, Eugene, OR,
USA). After three final washes in TSW, cells were resuspended in
phosphate-buffered saline (PBS)/glycerol supplemented with 0.1%
phenylendiammine, an antifade agent. Cells were visualized with
a Leica SP2 AOBS confocal laser scanning microscope equipped
with a 633 oil immersion objective. Alexa Fluor 488 and Alexa
Fluor 568 were detected at 488 nm excitation/520 nm emmission,
and 543 nm excitation/616 nm emission, respectively. The thickness
of the optical sections is specified in the legend of Fig. 3.
Leaves from young 4- to 6-week-old Nicotiana tabacum (cv Petit
Havana SR1) were infiltrated with A. tumefaciens containing the
appropriate plasmid at an optical density of 0.03 as described
previously (Batoko et al., 2000). Leaves were incubated for 2–3 d
at 25 C in daylight before observation. Small sections of infiltrated
leaves were placed on a microscope slide using double-sided tape
and visualized without a coverslip with a 633 water immersion objective attached to a Leica SP2 AOBS confocal laser scanning microscope. Simultaneous visualization of GFP, YFP, and chlorophyll
autofluorescence was performed using the sequential scanning facility of the microscope. Photomultiplier gain and offset were optimized to maximize each detector’s dynamic range using the ‘glow
over and under’ lookup table within the SP2 confocal acquisition
software. Figure compositions were assembled using Adobe Photoshop 6.0.
polar region only, with the substitution of the C-terminal
negative polar region of ER b5 with two arginines
(Borgese et al., 2001). To detect the proteins by immunofluorescence and biochemical techniques, tagged versions
of these sequences, termed b5-Nglyc and b5-RRNglyc,
were used in which an epitope containing an N-glycosylation
consensus sequence (OP3 epitope), recognized by a specific anti-opsin mAb (Adamus et al., 1991), was appended
at the C-terminus (Pedrazzini et al., 2000). The two
proteins were distributed either in the ER (b5-Nglyc) or in
the MOM (b5-RRNglyc) when expressed in mammalian
cells (Borgese et al., 2001). In an in vitro translation–
translocation system, both forms were instead posttranslationally translocated into dog pancreas microsomes,
indicating that the MOM isoform can be inserted into the
ER as well, and that unknown, additional factors present
in intact cells are required for correct targeting to the
MOM (Borgese et al., 2001).
We first investigated whether the two proteins are also
able to translocate their C-termini across plant microsomes
in vitro. mRNAs coding for either b5-Nglyc or b5-RRNglyc
were translated in the reticulocyte lysate system in the
presence of [35S]methionine. After stopping translation,
equal amounts of the labelled translation products were
incubated either in the absence or in the presence of microsomes purified from P. vulgaris developing cotyledons
(Lupattelli et al., 1997) for 1 h at 25 C. After incubation,
samples were analysed by SDS–PAGE and autoradiography. N-glycosylation affects migration of the protein in
SDS–PAGE, leading to a higher apparent molecular mass,
and this was used to assess the occurrence and efficiency
of translocation. Figure 1 shows that both proteins are
post-translationally glycosylated by plant microsomes: the
shift of b5-RRNglyc and b5-Nglyc to a higher molecular
mass form (asterisk in lanes 2 and 4) indicates the occurrence of glycosylation. After 1 h in the presence of
Results
Two mammalian isoforms of cytochrome b5 maintain
a different localization in tobacco protoplasts
Comparison of the C-terminal polar sequences of cyt b5
from plants and mammals showed that a net negative
charge is present in the mammalian ER isoforms only
(Borgese et al., 2001). The known cyt b5 sequences of
plants have C-termini with positive or neutral charge. It is
therefore possible that plants and mammals use different
mechanisms to sort and target cyt b5.
To address this question, it was first determined whether
two mammalian forms of cyt b5, resident either in the ER
or in the MOM, maintained their correct intracellular
locations once expressed in plant cells. The difference between the two forms is in the net charge of the C-terminal
Fig. 1. ER and MOM mammalian cytochrome b5 isoforms translocate
their C-terminus into plant microsomes in vitro. mRNAs encoding b5RRNglyc (lanes 1 and 2) or b5-Nglyc (lanes 3 and 4) were translated
for 1 h in the rabbit reticulocyte system in the presence of [35S]methionine. An equal amount of each protein was then divided into two
aliquots and incubated for 1 h in the absence (lanes 1 and 3) or presence
(lanes 2 and 4) of P. vulgaris microsomes (PhasMR). Samples were
analysed by SDS–PAGE/fluorography. Asterisks indicate glycosylated
polypeptides. On the left, the position of the 21 kDa marker is
indicated.
Sorting of tail-anchored proteins in plants 1369
microsomes, the glycosylated molecules represented 52%
of b5-Nglyc but only 23% of b5-RRNglyc, indicating that
b5-RRNglyc was glycosylated with lower efficiency than
b5-Nglyc (compare lanes 2 and 4). This is in contrast to
the results obtained by Borgese et al. (2001): in that study,
using dog pancreas microsomes, b5-RRNglyc was more
efficiently glycosylated than b5-Nglyc. This could reflect
some difference in the make-up of plant and mammalian
microsomes.
Having established that b5-Nglyc and b5-RRNglyc can
be targeted to plant microsomal membranes in vitro, the
destiny of the two proteins in living plant cells was investigated. Plasmids encoding b5-Nglyc and b5-RRNglyc
under the control of the CaMV 35S constitutive promoter
were transfected into tobacco mesophyll protoplasts.
Equal amounts of total protoplast homogenates were then
subjected to Endo H digestion before immunoblotting
with anti-opsin antibodies. As shown in Fig. 2, lane 3, two
polypeptides, with apparent molecular masses of 22 kDa
and 18 kDa, were detected when b5-Nglyc was expressed.
The 22 kDa polypeptide (Fig. 2, lane 3, asterisk) was entirely sensitive to digestion with Endo H, and the change
in mobility indicated that it represented the glycosylated
form of the 18 kDa polypeptide (compare lanes 3 and 4).
Therefore, the C-terminal polar sequence of b5-Nglyc is
efficently translocated and glycosylated in vivo. Conversely,
only a very minor proportion of b5-RRNglyc was glycosylated (Fig. 2, lanes 5 and 6). The results shown in Fig. 2
suggest that b5-Nglyc, but not b5-RRNglyc, is efficiently
targeted to the plant ER in vivo.
To determine the subcellular localization of the two
proteins, protoplasts were analysed by immunofluorescence
and confocal microscopy, using anti-OP3 mAb alone or in
combination with anti-BiP polyclonal antiserum. As shown
in Fig. 3A, b5-Nglyc showed a typical ER staining, which
Fig. 2. b5-Nglyc but not b5-RRNglyc is N-glycosylated in vivo.
Tobacco leaf protoplasts were transiently transfected with plasmids
encoding b5-Nglyc (lanes 3 and 4), b5-RRNglyc (lanes 5 and 6), or
with empty vector (lanes 1 and 2). At 24 h after transfection, cells were
lysed. Equal aliquots of each lysate were incubated with Endo H (lanes
2, 4, and 6) or without enzyme as control (lanes 1, 3, and 5). Samples
were analysed by SDS-PAGE followed by protein blot using anti-OP3
monoclonal antibodies. The asterisk in lane 3 indicates the position of
glycosylated b5-Nglyc. Numbers on the left indicate the position and
size (in kDa) of molecular mass markers.
is located in the interspace between chloroplasts (Fig 3B
and A/B merge in C), with the nuclear envelope clearly
stained as well (arrows). Moreover, upon double staining
with anti-OP3 and anti-BiP (Fig. 3G–I), the pattern of fluorescence of b5-Nglyc (Fig. 3G) was completely superimposed on that of the ER marker BiP (Fig. 3H and G/H
merge in I; yellow colour indicates co-localization). By
contrast, b5-RRNglyc (Fig. 3D) showed a distribution
which was superimposed to the envelope of chloroplasts
(Fig. 3E and D/E merge in F). Thus, we conclude that the
substitution of the C-terminal residues of ER b5 with
a pair of basic residues is sufficient to relocate the protein
from the ER to another membrane in plants as well as in
mammalian cells (Borgese et al., 2001), even if the target
membrane is not the MOM, but the chloroplast envelope.
Moreover, notwithstanding that, to date, plant cyt b5s with
a negative C-terminus have not been identified, b5-Nglyc
(that has a negative C-terminus) is targeted to the ER in
plant cells.
An in-depth search of the native Arabidopsis cyt b5
sequences was therefore performed to identify isoforms
with negatively charged luminal tails.
The Arabidopsis genome encodes six putative
isoforms of cytochrome b5
Within the A. thaliana proteome five sequences were identified (coded by the accessions At5g53560, At5g48810,
At2g46650, At1g26340, and At2g32720), and are listed
and aligned in Fig. 4. The primary structures of these
polypeptides are very similar and have the canonical
haem-binding site in the first half of the molecule; they
have a hydrophobic putative membrane-spanning domain
very close to the C-terminus, indicating a predicted TA
topology. Three of the identified proteins have a single
positive net charge in the short C-terminal polar sequence,
whereas At5g48810 and At5g53560 have a double positive
charge (Fig. 5A). No sequences with negatively charged
C-terminal luminal tails could be identified. Three isoforms, At2g32720, At2g46650, and At5g48810, have
more similarity to the identified ER isoform At5g53560
(Fukuchi-Mizutani et al., 1999). The putative cyt b5 coded
by the At1g26340 gene has a number of differences with
respect to the other four isoforms: its hydrophobic domain
is longer and has a distinct amino acid composition (Fig.
5A). This determines a lower average hydrophobicity and
lower maximal amphiphilicity of the a-helix (see Fig. 5B)
(analysis performed by ARAMEMNON transmembrane
domain topology and hydrophobic profile http://aramemnon.
botanik.uni-koeln.de/; Schwacke et al., 2003). This also
results in a different hydrophobicity profile of the tail
anchor of At1g26340 with respect to the other members
(Fig. 5C: only At1g26340 and At5g48810 are shown; the
other four members have characteristics very similar to the
latter). Moreover, the C-terminal polar region of At1g26340
has a net charge of +1, whereas that of At5g48810 has +2.
1370 Maggio et al.
Fig. 3. b5-Nglyc and b5-RRNglyc have a different intracellular localization in plant cells. Tobacco leaf protoplasts were transiently transfected with
plasmids encoding b5-Nglyc (A–C and G–I) or b5-RRNglyc (D–F). At 24 h after transfection, protoplasts were fixed, permeabilized, and analysed by
immunofluorescence microscopy, using anti-OP3 monoclonal antibodies alone (A–F) or in combination with a rabbit anti-BiP antiserum (G–I). AntiOP3 was detected with a secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (A, D, G), while anti-BiP was detected
with a secondary FITC-conjugated goat anti-rabbit antibody (H). Cells were observed with a confocal laser scanning microscope. (A–C) Merge of
50 Z-steps of 0.7 lm each; (D–F) merge of 55 Z-steps with a thickness of 0.84 lm each. (G, H) Single confocal section with a thickness of 1.8 lm.
(B, E) Chlorophyll autofluorescence. (C) A/B merge; (F) D/E merge; (I) G/H merge. Arrows indicate the nuclear envelope. Bar: 5 lm.
Based on these differences, it was decided to compare in
detail the intracellular fate of At1g26340 and At5g48810.
The polypeptides coded by the At5g48810 and At1g26340
genes are referred to as At5g and At1g, respectively.
At5g and At1g have a different intracellular localization
It was first tested if At5g and At1g polypeptides were able
to translocate their C-termini across plant ER microsomes
in vitro. To do this, the OP3 epitope was fused to the Ctermini of both the Arabidopsis cyt b5 sequences to detect
glycosylation. The two tagged polypeptides were called
At1g-Nglyc and At5g-Nglyc. The mRNAs coding for
either At1g-Nglyc or At5g-Nglyc were translated in the
reticulocyte lysate system and, after stopping translation,
equal amounts of 35S-labelled translation products were
incubated in either the absence or presence of P. vulgaris
microsomes for 1 h at 25 C. After incubation, samples
were analysed by SDS-PAGE and autoradiography. Figure
6A shows that both proteins can be post-translationally
glycosylated by plant microsomes: the shift of At1g-Nglyc
and At5g-Nglyc to a higher molecular mass form (asterisk
in Fig. 6A, lanes 2 and 4) indicated the occurrence of
glycosylation. Both proteins seemed to be glycosylated
with the same efficiency (;40%). It is concluded that
both proteins were able to translocate their C-termini posttranslationally across plant microsomes in vitro.
To investigate the intracellular destiny of At5g and
At1g, GFP or YFP were fused to their N-termini. The
fusion constructs, named YFP/GFP::At5g and YFP/
GFP::At1g, were subcloned into the vector pDHA (Tabe
et al., 1994) for transient expression in tobacco protoplasts, or into the vector pGreenII (Hellens et al., 2000)
for transient transfection of tobacco leaves mediated by
agroinfiltration.
Sorting of tail-anchored proteins in plants 1371
Fig. 4. Comparison of putative isoforms of cytochrome b5. Alignment of Arabidopsis cyt b5 isoforms showing the presence of conserved features of
the cyt b5 protein family, including the predicted haem-binding domain (residues 4–86) and C-terminal tail anchor (underlined). Identical residues
in all five isoforms are in white on a black background; blocks of similar residues are in black on a dark grey background; conserved substitutions are
in black on a light grey background; and non-similar residues are in black on a white background. Haem–iron ligands His40 and His86 are shown in
bold (Cowley et al., 2002). Sequences were aligned using the AlignX module, part of the Vector NTI advance (Invitrogen Corporation, CA, USA).
The expression levels and stability of the fusion proteins
were first determined by pulse–chase analysis. Protoplasts
from tobacco leaves were transiently transfected with
pDHA-GFP::At1g or pDHA-GFP::At5g and labelled with
[35S]methioine/cysteine for 2 h, followed by 3 h of chase.
At 0 h or 3 h of chase, cells were lysed and subjected to
immunoprecipitation with anti-GFP antibodies (Fig. 6B).
The two fusion proteins have the expected molecular
masses and do not undergo degradation (Fig. 6B, lanes 3
and 4, and 5 and 6). Very similar results were obtained
when the YFP::At5g and YFP::At1g constructs were
expressed (data not shown); GFP or YFP constructs were
therefore used depending on which was more convenient.
The intracellular localization of YFP::At5g was determined by confocal microscopy. Tobacco leaves were
infiltrated with a mixture of Agrobacterium strains transformed with pGreen-YFP::At5g and pBIN-GFP-HDEL.
The latter is a modified GFP, containing the HDEL
sequence for retrieval into the ER, commonly used as a
marker for this compartment (Haseloff et al., 1997).
Samples were analysed 3 d after infiltration. YFP::At5g
co-localized with GFP–HDEL, indicating that it is an ER
resident (Fig. 7A, B, and merge in C, yellow).
A similar co-infiltration experiment was performed
using a mixture of Agrobacterium strains transformed with
pGreen-YFP::At1g26340 or pBIN-GFP-HDEL. While GFP–
HDEL showed the typical reticular pattern (Fig. 8A),
YFP::At1g was instead located in close proximity to
chloroplasts (Fig. 8B), which could be identified by
chlorophyll autofluorescence (showed in Fig. 8C, blue). The
merge between the fluorescence given by GFP–HDEL,
YFP::At1g, and chloroplasts shows that YFP::At1g was
distributed around the chloroplasts, partially superimposed
on these organelles (Fig. 8D, violet), and did not colocalize with the ER marker. YFP::At1g was also present
in tubular structures (Fig. 8E, red), which interconnected
chloroplasts (Fig. 8F, blue, and E/F merge in G). These
structures, termed stromules, were previously observed in
several plants as highly dynamic stroma-filled tubules,
extending from the surface of all plastid types examined so
far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts (Natesan et al., 2005).
The results shown in Fig. 8 suggest that YFP::At1g may
be located on the COE. We therefore subcloned an expression cassette encoding the COE marker OEP7::GFP
(Lee et al., 2001) into pGreen II and infiltrated tobacco
leaves with a mixture of Agrobacterium strains transformed with pGreen-YFP::At1g or pGreen-OEP7::GFP.
Three days after infiltration, it was observed that the
pattern of distribution of YFP::At1g (Fig. 9B, red) was
completely superimposed on that of OEP7::GFP (Fig. 9A,
green) and that both proteins were located on the surface
of chloroplasts (Fig. 9C, blue, and A/B/C merge in D,
yellow). The two proteins were also located on stromules
(Fig. 9A, B, and D, asterisks), which were not detectable
by chloroplast intrinsic autofluorescence. From these
results, it was concluded that YFP::At1g is anchored to
the COE.
To determine whether a proportion of YFP::At1g was
also present on mitochondria, the marker mit-GFP, a GFP
targeted to the mitochondria by the signal sequence of the
b-ATPase subunit, was used (Logan et al., 2000).
Tobacco leaves were infliltrated with a mixture of Agrobacterium strains transformed with pGreen-YFP::At1g26340
1372 Maggio et al.
Fig. 5. Comparison of the C-terminal anchoring peptides of Arabidopsis cytochrome b5 isoforms. (A) Alignment of the transmembrane domains
(in grey text) of Arabidopsis cytochrome b5 isoforms, analysed by the THMM prediction algorithm; the C-terminal polar regions are in black and the
net charge of the C-terminal polar region is indicated. (B) The start and the end of each transmembrane domain, average hydrophobicity (HyPhob),
and maximal amphiphilicity calculated with ConPred_v2 (Arai et al., 2004). (C) Comparison between the hydrophobic profiles of At1g26340 and
At5g48810. The C-terminal anchoring region is boxed in grey.
or pGreen-mit-GFP. The expression level of YFP::At1g
was higher than that of mit-GFP; to obtain similar fluorescence levels, leaves were therefore infiltrated with an
unequal mixture of the two Agrobacterium strains. Two
different patterns of fluorescence were observed, which were
related to the presence or absence of chloroplasts: in cells
in which chloroplasts were detectable (Fig. 10A, blue),
YFP::At1g (Fig. 10C, red) never co-localized with mitochondria (Fig. 10B, green) and was always associated with
chloroplasts (Fig. 10D, merge of A/B/C, violet). By contrast, when chloroplasts were not detectable (Fig. 10E),
YFP::At1g (Fig. 10G, red) co-localized with mitochondria
(Fig. 10F, green and E/F/G merge in H).
This unexpected result indicates that YFP::At1g has the
capacity to localize promiscuously to both chloroplasts
and mitochondria, with a preference for the former.
To determine whether the different localizations of
YFP::At1g and YFP::At5g could also be detected
by subcellular fractionation, tobacco protoplasts were
transiently transfected with plasmids expressing either of
the two YFP fusions. All batches of protoplasts were also
co-transfected with plasmid expressing the marker of the
chloroplast envelope OEP7::GFP. At 24 h after transfection, protoplasts were homogenized in sorbitol buffer in
the absence of detergents, to maintain the integrity of the
organelles. The homogenates were then fractionated by
differential centrifugation: two fractions were isolated containing, respectively, particles sedimenting at 500 g (P1)
and 100 000 g (P2), and a third fraction containing the
100 000 g supernatant (PNS2). An equal amount of total
protein from each fraction was analysed by SDS-PAGE
and protein blot, using antibodies against GFP (which
Sorting of tail-anchored proteins in plants 1373
recognize YFP as well) and the ER marker BiP. As shown
in Fig. 11, BiP and OEP7::GFP were enriched in the P2
and P1 fractions, respectively. YFP::At5g was highly enriched in the P2 fraction whereas YFP::At1g was present
in both P1 and P2 fractions, with a slight enrichment in
the former. These results are largely consistent with the
localizations indicated by fluorescence; the presence of
a minor but relevant proportion of YFP::At1g in the P2
fraction could reflect enrichment of stromules in the
100 000 g pellet with respect to the 500 g pellet. Alternatively, this proportion of YFP::At1g could be localized on
the mitochondria, which often contaminate the 100 000 g
microsomal fraction. These results were also confirmed by
the expression of the two OP3-tagged form At5g-Nglyc
and At1g-Nglyc in living tobacco protoplasts: while the
first was totally glycosylated, the second never acquired
the glycan (data not shown).
Discussion
Fig. 6. At1g-Nglyc and At5g-Nglyc translocate their C-termini into
plant microsomal membranes in vitro and do not undergo rapid
degradation in vivo. (A) Synthetic RNAs coding for At1g-Nglyc (lanes
1 and 2) or At5g-Nglyc (lanes 3 and 4) were translated for 1 h in the
rabbit reticulocyte system in the presence of [35S]methionine. Each
sample was then divided into two aliquots and incubated for 1 h in the
presence (lanes 2 and 4) or absence (lanes 1 and 3) of P. vulgaris
microsomes (PhasMR). Samples were analysed by SDS-PAGE and
fluorography. Asterisks indicate N-glycosylated polypeptides. (B)
Tobacco leaf protoplasts transiently transfected with GFP::At1g (lanes
3 and 4), GFP::At5g (lanes 5 and 6), or mock-transfected (lanes 1 and
2) were pulse-labelled for 1 h with [35S]methionine and [35S]cysteine,
and subjected to chase for 0 h or 3 h. Cells were homogenized,
subjected to immunoprecipitation with anti-GFP antiserum, and analysed by SDS-PAGE and fluorography. Numbers on the left indicate
the position and size (in kDa) of molecular mass markers.
The mechanism of sorting and targeting of TA proteins
has in recent years been the object of research and debate,
but it is in most part still unclear. In mammalian cells, the
determinants of the intracellular sorting of the TA model
protein cyt b5 are concentrated in the short polar sequence
downstream of the transmembrane domain (Mitoma et al.,
1992; Kuroda et al., 1998; Borgese et al., 2001). In particular, the sorting of the two mammalian cyt b5 isoforms,
OMb5 and ERb5, is determined by the charge balance in
that region (Kuroda et al., 1998; Borgese et al., 2001).
Analysis of the sequence of the TA protein cyt b5 from
several different species revealed that the net negative
charge in the extreme C-terminal portion, characteristic of
the mammalian ER isoform, is not present in b5s of most
other organisms (Borgese et al., 2001). By contrast, known
cyt b5s of invertebrates, plants, and fungi generally have
a neutral or net positive charge in the C-terminal polar
domain. Consistently, we have identified five A. thaliana
cyt b5 putative isoforms, all characterized by a positive Cterminal end (+1 or +2, as shown in Fig. 5). Recently it
has been shown that the tung (Aleurites fordii) cyt b5
isoforms A, B, and C, and the cauliflower cyt b5 are
located on the ER (Zhao et al., 2003; Hwang et al.,
2004). It is therefore possible that the charge-based
Fig. 7. YFP::At5g is sorted to the ER. Confocal microscopy of tobacco leaves infiltrated with a mix of Agrobacterium strains carrying the ER marker
GFP-HDEL (A) or YFP::At5g (B). Simultaneous visualization of GFP and YFP from the same field was performed using the sequential scanning
facility of the microscope. (C) A/B merge. Bar: 20 lm.
1374 Maggio et al.
Fig. 9. YFP::At1g is targeted to the chloroplast envelope. Tobacco
leaves were infiltrated with a mix of Agrobacterium strains carrying the
chloroplast envelope marker OEP7::GFP or YFP::At1g, and analysed
by confocal microscopy. (A) GFP; (B) YFP; (C) chlorophyll autofluorescence; (D) A/B/C merge. Asterisks indicate stromules. Bar: 4 lm.
Fig. 8. YFP::At1g is localized around chloroplasts and on the
stromules. Tobacco leaves were infiltrated with a mix of Agrobacterium
strains carrying the ER marker GFP-HDEL or YFP::At1g and analysed
by confocal microscopy. (A) GFP; (B) YFP; (C) chlorophyll autofluorescence; (D) A/B/C merge. A particular of a second field is shown in E
(YFP), F (chlorophyll autofluorescence), and G (E/F merge). Asterisks
indicate stromules that connect the two chloroplast envelopes. Bar:
4 lm.
discrimination between MOM and ER does not operate, or
operates at lower efficiency, in plants. To shed light on this
question, the in vitro and in vivo subcellular targeting of
plant and mammalian TA proteins in plant cells was
investigated.
Targeting to the ER
Cyt b5 has been considered an ER protein in plants
(Smith et al., 1994), but recent studies indicate the presence of isoforms targeted to other compartments (Zhao
et al., 2003; Hwang et al., 2004). Here it was first verified
whether two mammalian cyt b5 forms, b5-Nglyc and b5RRNglyc, located on the ER membrane and MOM in mammalian cells, maintain the same locations in a plant cell.
Using transient expression in tobacco protoplasts, the ER
localization of b5-Nglyc was also confirmed in a plant
cell, but the mammalian MOM form was sorted predom-
inantly to the chloroplast envelope. The conserved ER
targeting in plants and mammals indicates that, although
all plant cytochromes have a positive C-terminus, a negative net charge is recognized in plant cells as an ERtargeting sequence, strongly suggesting that the still poorly
characterized mammalian ER targeting mechanism also
exists in plants.
The present in vitro experiments, however, indicate that
there might be differences between plants and mammals
in the charge-based sorting mechanism. Previous
experiments (Borgese et al., 2001) showed that the MOM
b5-RRNglyc form can be translocated in vitro into mammalian microsomes, indicating that in vivo MOM targeting
operates at least in part through a dominant ER-targeting
avoidance mechanism. In vitro, the MOM form is indeed
more efficiently glycosylated than the ER form b5-Nglyc
by mammalian microsomes. It is shown here that in
in vitro experiments the MOM form is also translocated
into plant microsomes, but its translocation efficiency is
lower than that of the ER form. Thus, in vitro translocation
is more representative of the in vivo process in plant than in
mammalian cells. Clearly the differences between microsomes purified from plants and mammals require further
elucidation.
Having shown by expression of mammalian proteins
that a charged-based mechanism also exists in plant cells,
Sorting of tail-anchored proteins in plants 1375
Fig. 11. Subcellular distribution of YFP::At5g and YFP::At1g investigated by subcellular fractionation. Leaf tobacco protoplasts, transiently
transfected with YFP::At5g, YFP::At1g, or OEP7::GFP cDNAs, were
lysed and subjected to subcellular fractionation by differential centrifugation. The first fraction was recovered by centrifuging the lysate at
500 g (P1). The resulting supernatant was collected and centrifuged
again at 100 000 g: the pellet (P2) contained the microsomal and
mitochondrial fractions and the supernatant (PNS2) contained the
cytosolic and vacuolar soluble proteins. The distribution of YFP::At5g
and YFP::At1g was analysed by SDS-PAGE and protein blotting using
anti-GFP antiserum (middle panels) and compared with that of a marker
for the ER (anti-BiP antiserum, top panel) and the chloroplast envelope
marker OEP7::GFP (anti-GFP antiserum, bottom panel). Numbers on
the left indicate the position and size (kDa) of molecular mass
markers.
Fig. 10. YFP::At1g is able to reach both the chloroplast envelope and
the mitochondria, but is preferentially localized on the chloroplasts.
Two different fields of tobacco leaves were infiltrated with a mix
of Agrobacterium strains carrying the mitochondrial marker mit-GFP
(B, F, green) or YFP::At1g (C, G, red). Photosynthetic (A–D) or
non-photosynthetic (E–H) cells were visualized by confocal microscopy.
(A, E) Chlorophyll autofluorescence; (B, F) GFP; (C and G) YFP; (D)
A/B/C merge; (H) E/F/G merge. Bars A–D, 8 lm; E–H, 16 lm.
an investigation was begun to determine if another sorting
mechanism operates on the different Arabidopsis cyt b5
isoforms. Two A. thaliana cyt b5 isoforms, selected to
have the most divergent features within domains that are
presumably involved in targeting, were used. Using both
biochemical and morphological approaches, the intracellular localization of At1g and At5g proteins was determined
in transiently transfected tobacco protoplasts and in agroinfiltrated epidermal tobacco cells. Notwithstanding its
positive charge in the C-terminal polar sequence, At5g is
definitely localized on the ER membrane. Altogether, the
results of investigations of expression of mammalian and
plant cyt b5 forms indicate that plant ER is less selective
than mammalian ER with regard to TA protein targeting,
because, apparently, it is able to accommodate cyt b5
forms with opposite charges.
Targeting to mitochondria and chloroplasts
The subcellular localization of plant cyt b5 isoforms is
controversial. Zhao et al. (2003) showed that in cauliflower a single cyt b5 is able to reach both the ER and the
mitochondrial membrane. Recently, Hwang et al. identified four isoforms of cyt b5 in tung. They concluded that
three of them are located on the ER (cyt A, B, and C) and
one (cyt D) on the mitochondria (Hwang et al., 2004).
The mitochondrial form is very similar to the At1g
1376 Maggio et al.
isoform, with the C-terminal polar sequence RKK instead
of RKT (see At1g26340 in Fig. 5A). The authors also
constructed a chimera by substituting the C-terminal polar
residues of tung cyt b5 D with the polar sequence of At1g,
and concluded, in contrast to the present results, that the
Arabidopsis isoform is also targeted only to mitochondria.
These contrasting results are possibly due to the use of a
chimera, with the bulk of tung protein and only three
amino acids from the Arabidopsis polypeptides, whereas
the native At1g protein was used in the present study.
Moreover, the study by Hwang et al. (2004) was
performed in BY2 tobacco suspension cells, whereas
agroinfiltrated tobacco leaves or protoplasts were used
here. It is possible that in BY2 cells, which have nonphotosynthetic undifferentiated proplastids—whose functions differ from the major activities of chloroplasts
(Baginsky et al., 2004)—the tung/Arabidopsis chimera
fails to be sorted to these organelles and is mislocalized
to the mitochondria (this would point to a competition
between chloroplasts and mitochondria for the sorting of
At1g). Alternatively, At1g could be sorted in BY2 cells to
proplastids which are too small to be distinguished from
mitochondria by non-confocal fluorescence microscopy.
The leaf agroinfiltration expression system provides a snapshot of TA targeting which reflects reliably cyt b5 sorting
events in differentiated leaf tissues. Thus both chloroplast
localization and, in the absence of these organelles,
mitochondrial localization could be highlighted. Zhao
et al. (2003) showed that in cauliflower a single cyt b5
was localized both on the ER and in mitochondrial
membranes in onion epidermal cells. It cannot be ruled
out that the cauliflower cyt b5 would also be targeted to
chloroplasts in cells with autotrophic plastids. Another TA
protein, Bcl-2, is localized at both ER and mitochondria in
mammals and has slightly different functions at each location (Zhu et al., 1996; Kim et al., 1997). Moreover, the
MOM form of cytochrome b5 is able to be targeted to
microsomes in vitro, but in vivo the MOM wins the competition with the ER to capture this molecule (Borgese
et al., 2001). It can be concluded that the localization of
cyt b5 isoforms may depend on both cell type and cell
function. The identification of the molecular mechanism
involved in sorting of At1g to COE or MOM will shed
light on this.
Similarly to At1g, several small proteins encoded by the
nuclear genome and resident on the COE do not have
a cleavable targeting signal and do not use a general
import pathway (Salomon et al., 1990; Wu and Ko, 1993;
Li and Chen, 1996). The outer membrane proteins (OEPs)
that lack the classic chloroplast transit peptide, such as
spinach OEP6.7, pea OEP14, pea OEP34, and AtOEP7,
were targeted to purified chloroplasts when assayed in an
in vitro import system (Salomon et al., 1990; Wu and
Ko, 1993; Li and Chen, 1996) and in vivo in protoplasts
or in transgenic plants (Lee et al., 2001). OEPs have an N-
terminal targeting signal, containing a hydrophobic stretch
(Lee et al., 1991; Wu and Ko, 1993; Li and Chen, 1996)
and the bulk of the polypeptide results embedded in the
cytosol; the topology of these proteins could therefore be
defined as ‘head-anchored’. The function of all of these
OEPs is not known, but they could have a role in chloroplast biogenesis and homeostasis. Another protein without a cleavable transit peptide, but with a TA topology, is
the import receptor Toc34 (Seedorf et al., 1995; Qbadou
et al., 2003). This protein also has a role in chloroplast
biogenesis. Toc34 can be inserted into purified chloroplasts, but not into mitochondria (Qbadou et al., 2003).
Moreover, the protein is also able to insert into protein
free-liposomes, and the integration process, stimulated by
GTP, depends on the primary sequence and tertiary
structure of Toc34, as well as the lipid composition of the
target membrane. It is not yet clear whether ER targeting
of cyt b5 requires specific helper or receptor proteins. The
C-terminal polar sequence of the tung ER isoforms Cb5A
and Cb5B prevents spontaneous insertion of these proteins
in other membranes, indicating that the recognition of
these sequences by the ER but not by the mitochondrial
insertion machinery results in a selective insertion into the
ER (Henderson et al., 2006), confirming and extending
previous results (Smith et al., 1994). However, the ER
isoform of mammalian cyt b5 is able to insert into proteinfree phospholipid vesicles or native ER microsomes with
similar efficiencies, and increasing cholesterol to levels
found in other membranes of the secretory pathway
inhibits its translocation, indicating that insertion depends
on lipid composition rather than specific protein machinery (Brambillasca et al., 2005). According to all these
data, it is reasonable to think that the ability to insert
directly in the lipid bilayer without the help of translocons
and molecular machinery could be a feature of ancestral
proteins which were able to recruit lipids, contributing to
the formation of inner membranes during the evolution of
subcellular compartmentalization and therefore to the
passage from prokaryotes to eukaryotes. Subsequently, or
at the same time, when different organelles were forming,
a finer mechanism of regulation of the TA protein sorting
occurred. Cyt b5, because of its role in lipid metabolism,
could have played a role in the formation of highly
dynamic membranes where lipids are mainly synthesized
(ER, COE, and MOM), whereas other proteins (such as
Sec61, Tom, Toc, and Pex) would have specialized in the
formation of the translocation channels.
What could be the function of cyt b5 on the COE?
Analysis of the chloroplast proteome has emphasized unexpected functions of envelope membranes, in which cyt
b5 could play a role (Ferro et al., 2003; Rolland et al.,
2003). It has been demostrated that cyt b5 directly modulates the activity of microsomal flavonoid 3#,5#-hydroxylase, a cytochrome P450 involved in the biosynthesis of the
anthocyanins, both in petunia flowers and in grapevine fruits
Sorting of tail-anchored proteins in plants 1377
(de Vetten et al., 1999; Bogs et al., 2006). Cyt b5 and its
reductase catalyse the microsomal reduction of xenobiotic
hydroxylamines and amidoximes in humans (Kurian et al.,
2004). A number of cytochrome P450 family members,
such as tomato hydroperoxide lyase (HPL; Froehlich
et al., 2001), pea CYP86B1 (Watson et al., 2001),
Arabidopsis ent-kaurene oxidase (AtKO1; Helliwell et al.,
2001), allene oxide synthase, and a putative amine oxidase (CP74 and HP52b, respectively; Rolland et al., 2003)
are localized on the chloroplast envelope, suggesting possible roles for cyt b5 on COE. Finally, an electron transfer chain exists on the chloroplast envelope, involved in
fatty acid desaturation and lipid metabolism (Jager-Vottero
et al., 1997; Rolland et al., 2003).
Do plant cytochrome b5 proteins use the
charge-based sorting mechanism?
It has been shown that the charge-based sorting mechanism
operates in plant cells, but it remains to be demonstrated
that wild-type plant proteins use it. Based on the observation that two TA proteins with positive charge at their
C-terminus are sorted to different membranes, it could be
concluded that TA protein sorting in plants is not based
on the net charge of the C-terminal polar sequence. However, analysing the C-terminus of the ER-located isoform
At5g using Net-Phos 2.0 prediction server (http://www.cbs.
dtu.dk/services/NetPhos/), it was found that it contains a
number of putative phosphorylation sites (in serine, threonine, and tyrosine; see Supplementary Fig. S1 at JXB
online).
Phosphorylation would revert the net charge from positive to negative. Such a mechanism would also have
regulatory implications, because the cell could modulate
the localization of a TA protein by changing its Cterminal charge by phosphorylation. This possibility is
currently being investigated and the C-termini of other
plant ER TA proteins are being analysed in order to formulate a general model for sorting of these polypeptides.
Supplementary data
Analysis of the putative phosphorylation sites in the
At5g48810 sequence is shown in Supplementary Fig. S1
at JXB online. In particular, the sites that could be
phosphorylated in the C-terminal polar sequence and that
could be involved in sorting to the ER have been
highlighted.
Acknowledgements
We are grateful to Dr Paul Hargrave (University of Florida) for his
kind gift of hybridoma cells producing anti-opsin mAbs (R2-15)
against the N-terminal peptide of bovine opsin (R2-15), to Dr
Inhwan Hwang for kindly providing us with the OEP7::GFP fusion
construct, and to Dr David Logan (St Andrews University) for mitoGFP. We thank the Arabidopsis Biological Resource Center
(ABRC) DNA stock center for kindly providing the Arabidopsis
thaliana cytochrome b5 cDNAs At1g26340 and At5g48810. We are
particularly grateful to Dr Alessandro Vitale for helpful discussion
and for critically reading the manuscript. We would like to thank
Dr Nica Borgese for financial support at the beginning of this work
with a grant from Ministero Università e Ricerca (PRIN 2002). This
work was supported in part by grants from VI European FW
Programme Priority (LSH-2002-1.2.5-2) ‘Recombinant Pharmaceuticals from Plants for Human Health—Pharma-Planta’.
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