PsaK paper

1

M1:02914, revised version. July 7, 2001

Insertion of PsaK into the thylakoid membrane in a 'horse-shoe' conformation

occurs in the absence of signal recognition particle, nucleoside triphosphates or

functional Albino3

Alexandra Mant1*, Cheryl A. Woolhead1,2, Misty Moore3, Ralph Henry3, and Colin Robinson1

1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

2Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom

3Department of Biological Sciences, University of Arkansas, Fayetteville AR 72701

Corresponding author: Colin Robinson

Telephone: +44 2476 523557

Fax: +44 2476 523568

Email: [email protected]

*Present address

Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and

Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

Running title: Sec/SRP/Alb3-independent insertion of PsaK

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 12, 2001 as Manuscript M102914200 by guest on February 6, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

SUMMARY

The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N- and C-termini

both located in the lumen. The insertion mechanism of a thylakoid membrane protein adopting this

type of topology has not been studied before, and we have used in vitro assays to determine the

requirements for PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that

one transmembrane span (the C-terminal region) can insert independently of the other, indicating

that a 'hairpin'-type mechanism is not essential. Insertion of PsaK does not require stromal extract,

indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside

triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting

a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of

SRP or its partner protein, FtsY. We furthermore find no evidence for the involvement of known

membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat

translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog,

Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion.

Because YidC is required for the efficient insertion of every membrane protein tested in

Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as

being independent of YidC/Alb3-type factors in either thylakoids or bacteria. The data raise the

possibility of a wholly spontaneous insertion pathway.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

3

INTRODUCTION

Studies in bacteria and plant thylakoids have demonstrated the operation of a complex signal

recognition particle (SRP)-dependent pathway for the insertion of membrane proteins. This pathway

was first demonstrated in bacteria (reviewed in [1]) where the insertion of several plasma membrane

proteins was shown to require the activity of two soluble/extrinsic proteins, SRP and FtsY.

Escherichia coli SRP is a complex comprising a 4.5S RNA molecule together with a homolog of

the 54 kDa subunit of eukaryotic SRPs, which binds to particularly hydrophobic regions such as

nascent or newly-synthesized membrane proteins. The SRP-substrate complex interacts, at least in

some cases, with the SecYEG translocon in the plasma membrane and, in a poorly-understood

sequence of events, the substrate is transferred into the translocon with the assistance of an

additional factor, FtsY [2-8]. Both SRP and FtsY are GTPases and the insertion process depends

totally on GTP hydrolysis.

A broadly similar pathway has been characterised for the insertion of the major light-harvesting

chlorophyll-binding protein, Lhcb1, into the thylakoid membrane of plant chloroplasts. This

conservation of insertion pathway is perhaps not surprising, given that chloroplasts are widely

accepted to have evolved from endosymbiotic cyanobacterial-type organisms. After import from the

cytosol, Lhcb1 binds stromal SRP to form a soluble targeting complex [9]. Integration of the

polytopic Lhcb1 protein into the thylakoid membrane further requires FtsY [10,11], GTP and a

membrane-bound translocase. The translocase has yet to be fully characterized; antibodies to SecY

strongly inhibit the insertion of SecA-dependent lumenal substrates but do not block the insertion of

Lhcb1 [12,13]. This raises the possibility that the thylakoid SecYEG complex is not required,

although this work could not rule out the possibility that the SRP interacts with a different SecY

determinant that is unaffected by antibody binding. Interestingly, the chloroplast SRP particle

differs from that of E. coli in that (i) no RNA is present and (ii) this SRP possesses a novel 43 kDa

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

4

subunit [14], which binds to a novel SRP recognition element in Lhcb1 that is found in other

members of the light-harvesting chlorophyll a/b –binding protein family (LHC proteins) [15,16].

Nevertheless, there are clear parallels with the E. coli insertion pathway and the similar natures of

the pathways are reinforced by recent findings concerning the essential involvement of Oxa1-type

proteins. Oxa1 is a mitochondrial inner membrane protein that is involved in the biogenesis of a

range of mitochondrial inner membrane proteins, specifically those that insert from the matrix side

of the membrane [17, 18]. An Oxa1 homologue, termed Alb3, is also essential for the insertion of

Lhcb1 in thylakoids [13] and recent work has shown an E. coli homologue, YidC, to be essential for

the insertion of at least some SRP-dependent membrane proteins in this organism [19].

Critically, YidC is also essential for the biogenesis of E. coli membrane proteins that do not require

SRP or the Sec apparatus. The insertion mechanisms of M13 procoat and Pf3 coat protein have

been characterised in some detail [20] and shown to insert efficiently into the plasma membrane in

the complete absence of functional SRP, SecA or the membrane-bound SecYEG translocon. On the

basis of these data it had been suggested that these proteins may insert spontaneously into the

membrane, but depletion of YidC led to a rapid block in their insertion indicating a central role in

their insertion. Thus, it appears that one pool of YidC may be associated with the Sec apparatus [21]

while another pool may in effect represent a novel form of translocase dedicated for the insertion of

some, if not all, SRP-independent membrane proteins. Like M13 procoat, a subset of thylakoid

membrane proteins are synthesized with cleavable signal peptides but inserted in the absence of

SRP [22,23], but the possible involvement of the YidC homolog, Alb3, remains to be clarified.

All of the previous studies on thylakoid protein insertion have focused either on relatives of the

well-studied Lhcb1 (LHC proteins), or on proteins that bear cleavable N-terminal signal peptides. In

this report we have sought to characterise the insertion of a different type of thylakoid membrane

protein, PsaK, which is not synthesized with a cleavable signal peptide and which is unrelated to

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

5

LHC proteins. We show that this protein inserts with high efficiency into thylakoids by a

mechanism that does not involve SRP, NTP hydrolysis or any of the known translocation

machinery in the thylakoid membrane, including Alb3.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

6

EXPERIMENTAL PROCEDURES

DNA Constructs

A full-length cDNA clone encoding the precursor of barley PsaK (pPsaK) in the plasmid

pBluescriptSK(-) was a gift from B. L. Møller [24]. A construct encoding the mature PsaK

protein was prepared by polymerase chain reaction amplification of the aforementioned barley

cDNA. The forward primer (5’-CAG GGG ATC CGC ATG GAC TAC ATC GGC-3’) introduced a

BamH I site between bp 287 and 288, and altered cysteine 42, which is the last amino acid of the

transit peptide, to an initiating methionine residue. The reverse primer (5’-CCC GAA GCT TGC

AGA ACA GCT ATG-3’) introduced a Hind III restriction site between bp 603 and 604, after the

stop codon at bp 565. The amplified region was cloned 5’ BamH I-Hind III 3’ into pGEM4Z, and

sequenced completely before being used as a template for transcription and translation. A cDNA

clone encoding pea pLhcb1 was provided by N.E. Hoffman [25], while the cDNA clone encoding

wheat p23K has been described [26].

Transcription and Translation

The plasmid encoding pPsaK was linearized with Xho I, whereas linearization was found to be

unnecessary for the plasmid encoding PsaK. Transcription in vitro was performed according to

Promega protocols, using either T3 RNA polymerase (for pPsaK), or SP6 RNA polymerase (for

PsaK, pLhcb1 and p23K). Radiolabeled proteins were prepared using a wheat germ lysate system

(Promega) in the presence of [35S] methionine (Amersham Pharmacia), also according to the

manufacturers’ instructions. The translation mixtures were treated with puromycin and centrifuged

prior to use in insertion assays, as described in Thompson et al. [27].

Import assays

Assays for the import of precursor proteins by intact pea chloroplasts and isolated thylakoid

membranes were essentially as described in [28] except that the light intensity was 300 µmol

photons m-2 s-1. The proton ionophore nigericin (Sigma) was dissolved in ethanol, and used at a

final concentration of 2 µM in the presence of 10 mM KCl; control samples were identical, except

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

7

they contained an equivalent volume of ethanol instead of nigericin. Assays to measure the effect of

apyrase (Sigma, type VI) were carried out as described in [27]. 10 mM stock solutions of the non-

hydrolyzable ATP and GTP analogues, AMP-PNP and GMP-PNP (Sigma) were prepared in 10

mM Hepes, 5 mM MgCl2 and the pH adjusted to 7. When used, the final concentrations of these

analogues were 0.5 mM. Proteolysis of thylakoid membranes, prior to insertion assays, was as

described in [27] and Alb3 antibody-inhibition tests were as described in [13], except that the

thylakoids were incubated with anti-Alb3 antibodies for 2 h instead of 1 h.

Treatment of Thylakoid Membranes, Post Assay

In order to test if PsaK was correctly inserted in the thylakoid membrane (either after import into

intact chloroplasts, or after insertion into isolated thylakoid membranes), the membranes (between

10 and 20 µg chlorophyll, depending on the experiment) were washed with 0.5 ml ice-cold10 mM

Hepes-KOH pH 8, 5 mM MgCl2 (HM) and then reisolated by centrifugation at 18,000 g and 4°C for

5 min in a microcentrifuge. Next, they were washed with 100 µl 20 mM Tricine-NaOH, pH 8 (TB)

and reisolated as above, then subjected to one round of extraction with 6.8 M urea, using a protocol

adapted from Breyton et al. [28] and described in detail in [27]. Next, the membranes were

resuspended in TB and digested with 0.2 mg ml-1 trypsin (Sigma, type XIII) in a final volume of

100 µl for 30 min on ice. Trypsin digestions were stopped by the addition of 0.5 mg ml-1 trypsin

inhibitor (Sigma, type I-S), followed by centrifugation at 18,000 g and 4°C for 10 min in a

microcentrifuge. Finally, the thylakoid membranes were resuspended in 15 µl TB containing 5 µg

trypsin inhibitor, and an equal volume of 2 X protein sample buffer, then immediately boiled for 5

min. Insertion efficiencies were measured by exposing dried SDS-PAGE gels in phosphorimager

cassettes, followed by quantitation in a Molecular Dynamics PhosphorImager.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

8

RESULTS

Structures of the precursor and mature forms of PsaK

PsaK is a nuclear-encoded subunit of photosystem I that is important for the stable interaction of

LHCI with the photosystem I core complex (29). Barley PsaK is synthesized in the cytosol as a 13.7

kDa precursor, imported into the chloroplast and subsequently inserted into the thylakoid membrane

[24]. The presequence is a typical stroma-targeting peptide that is predicted to be removed by the

stromal processing peptidase (SPP). From sequence analysis, the 9 kDa mature protein is predicted

to span the membrane twice with the N- and C-termini both located in the lumen and the positively

charged loop region remaining on the stromal (cis) side of the thylakoid membrane according to the

‘positive-inside rule’ [30]. Recent high-resolution crystallographic analyses of Synechococcus

elongatus photosystem I have confirmed these predictions for the cyanobacterial PsaK, which is

highly similar to eukaryotic PsaK (P. Jordan, P. Fromme and N. Krauss, personal communication).

This 'horse-shoe' configuration is illustrated in Figure 1, which also indicates the locations of the

methionine residues in the mature barley protein (two in the first transmembrane region, one in the

second). The stroma-exposed loop region contains numerous positively-charged residues and

trypsin is therefore predicted to cleave in this region and generate two degradation fragments

containing the indicated numbers of methionine residues. The amino acid sequence of pPsaK is also

given in Fig. 1 together with the start site of a mature-size construct described below.

Insertion of PsaK does not require SRP or nucleoside triphosphates, but is stimulated by the ∆µH+

Figure 2 shows a chloroplast import experiment conducted with [35S]-methionine-labeled barley

pPsaK. The precursor protein (pPsaK) is imported into chloroplasts (lane C) where it is processed to

the mature form and resistant to added protease (lane C+); the mature protein has an apparent mass

of 7 kDa (in agreement with [24]) and is found exclusively in the thylakoid fraction (lane T). The

protein is highly resistant to urea extraction (lane Turea), which has been shown to be an effective

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

9

means of identifying many membrane proteins [28]. Trypsin-treatment of the thylakoids from lane

T (lane 1) leads to the generation of two fragments, one of which contains precisely twice as much

[35S] radioactivity as the other (data not shown, from Phosphorimager analysis). On the basis of the

data described above, the stronger, lower band most likely represents the first transmembrane

region (TM1) and the other (TM2) is the C-terminal transmembrane span. If the thylakoids are

washed with urea after trypsin-treatment, a proportion of the fragments are extracted (lane 2)

whereas the same fragments observed in lane 1 are obtained if the membranes are urea-treated prior

to proteolysis (lane 3). Clearly, mature PsaK is far more resistant to urea-extraction than either of

the single-span degradation fragments and we believe that this reflects the ability of urea to partially

extract small, single-span proteins, whereas proteins containing two or more spans are almost

totally resistant. We have observed a similar phenomenon with single-span proteins bearing signal

peptides: the precursor proteins adopt loop conformations in the membrane that are far more

resistant to urea extraction than the single-span mature proteins [27].

The primary aim in this work was to analyse the mechanism by which PsaK inserts into the

thylakoid membrane, and for these experiments we used in vitro assays for the insertion of

radiolabeled protein into purified pea thylakoids. Our criteria for correct insertion were (i) that the

protein should be resistant to urea extraction and (ii) that trypsin should generate the two

degradation products observed after import of pPsaK as shown in Fig. 2. Fig. 3A shows the results

of incubating the pPsaK translation product (lane Tr) with thylakoids in the presence of stromal

extract: the mature protein is efficiently generated by SPP in the extract, a large proportion of

mature PsaK becomes associated with the thylakoids (lane T) and urea-extraction followed by

trypsin digestion produces the two diagnostic degradation products (TM1 and TM2). These results

effectively confirm that insertion is taking place. However, the full precursor protein is unsuitable

for detailed tests on the insertion requirements because stromal extract (which contains essentially

all of the SRP) can not be omitted since it also contains almost all of the stromal peptidase required

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

10

to generate the mature form of PsaK. The N-terminus of mature PsaK is located in the lumen, which

means that processing to the mature size must precede insertion. We have found that the full

precursor form does not insert correctly in the absence of stromal extract (see below), which is

unsurprising because the entire presequence would have to be translocated across the thylakoid

membrane. Further experiments were therefore conducted with a mature-size PsaK translation

product synthesized from a truncated cDNA as described in Experimental Procedures.

Fig. 3B shows assays for the insertion of this construct (denoted PsaK) into isolated thylakoids.

After each assay, samples of the thylakoids were either treated with trypsin (upper panel) or urea-

extracted (lower panel). PsaK again inserts into isolated thylakoids and the inserted protein is again

converted to the diagnostic degradation products TM1 and TM2 ('+ trypsin' panel). As a control, we

incubated the translation product with trypsin in the absence of membranes (lane Tr+) and this

procedure leads to an almost complete degradation of PsaK, with no evidence for either the TM1 or

TM2 bands (providing further evidence that TM1 and TM2 represent membrane-integrated

regions). In this assay, TM1 should contain three times as much [35S]-Met as TM2 because of the

initiation methionine introduced during the cloning procedure, which is located in the lumen after

insertion and hence protected from proteolysis. In repeat assays such as those shown in Fig. 3B, the

TM1:TM2 35S-Met ratio was always near 3 (within 15% in each case) confirming correct insertion,

and this is supported by the observation that the mature protein is equally resistant to urea extraction

('+ urea' panel). We have routinely found that PsaK inserts with very high efficiency in this assay

system; 34% of translation product was found to be inserted in this particular experiment (based on

the recovery of 35S-Met in TM1 and TM2) and insertion efficiencies of over 40% have been

observed in other experiments (data not shown).

The left hand panel of Fig. 3B shows assays carried out in the presence of stromal extract (SE) or

Hepes-magnesium buffer (HM), and in each of these cases insertion was assayed either with or

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

11

without pretreatment of the assay mixture with apyrase (indicated as +/- above the lanes). This

enzyme hydrolyzes nucleoside triphosphates in the mixture and totally blocks insertion by the SRP

route [9,11]. These lanes show that insertion is most efficient in the HM incubations, i.e. in the

absence of stromal extract (and hence SRP). The presence of apyrase does, however, lead to a

reduction in insertion efficiency (to 71% of the control value) but this is not due to SRP

involvement because the presence of stromal extract also leads to a slight reduction in insertion

efficiency (down to 81% of the HM value). Stromal extract contains essentially all of the SRP (see

below) and this result rules out an SRP involvement.

In the same experiment we also assessed the effects of including GMP-PNP and AMP-PNP (non-

hydrolyzable analogs of GTP and ATP, respectively) in an attempt to identify the cause of the

reduction observed with apyrase. GMP-PNP, in particular, is an effective inhibitor of SRP [11].

However, neither analog inhibits insertion to any marked extent, either in the absence or presence of

stromal extract (right hand panel in Fig. 3B).

As a control for these experiments we used insertion assays with Lhcb1, a known substrate for the

SRP pathway. After insertion into thylakoids, Lhcb1 becomes highly resistant to trypsin digestion

and a characteristic stable degradation product is generated [9,11-13]. Fig. 3C shows that insertion

is completely dependent on stromal extract, as shown by the appearance of the degradation product

(DP) in the SE panel. Insertion is also completely dependent on the presence of NTPs in the mixture

as pretreatment with apyrase (lanes indicated by +) totally blocks insertion, as found previously

[9,11]. On the basis of these data we conclude that PsaK does not require SRP for its insertion and,

because insertion is not even stimulated by the presence of stroma, we propose that insertion is

indeed completely independent of SRP. Insertion is, however, slightly inhibited by apyrase

treatment even in the absence of stromal extract, which indicates a mild stimulatory influence of an

ATP or GTP-hydrolyzing factor in the wheatgerm translation system. We believe the most likely

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

12

explanation to be an anti-aggregation effect of chaperones in the wheatgerm extract which may

prevent some PsaK molecules from adopting an insertion-incompetent conformation.

The above studies rule out a central role for NTP hydrolysis in the insertion of PsaK but to obtain a

more comprehensive picture of the energy requirements we tested whether the thylakoidal proton

motive force stimulates insertion. Figure 4A shows the effect of nigericin (a proton ionophore) on

the import and sorting of pPsaK in intact chloroplasts. In the control assay, the protein is found

exclusively in the thylakoid fraction, and only as the mature form. With nigericin present, however,

some imported protein is found in the stromal fraction and it is notable that the primary stromal

form is the full precursor protein. Some mature PsaK is also found in the stroma, together with a

further, intermediate-size protein (iPsaK) that may represent a processing intermediate

(alternatively, this may result from proteolysis of pPsaK). From these data it is evident that optimal

insertion efficiency is dependent on the thylakoidal proton electrochemical gradient, ∆µH+. The

appearance of the full precursor protein furthermore suggests that pPsaK is not necessarily

processed immediately upon entry into the stroma, and we suggest in addition that at least some

molecules may be processed only during the later stages of the insertion pathway, since the

appearance of pPsaK most likely stems from an inhibition of insertion.

To obtain further data on the ∆µH+-dependence of insertion we conducted thylakoid insertion

assays with PsaK in the presence of nigericin (Fig. 4B). The data show that the presence of

nigericin (lanes N) reduces insertion efficiency to a moderate extent when compared with the

control samples shown in lanes C (in this experiment by 20-25%) in both the presence and absence

of stromal extract. Taken together, these data indicate that the ∆µH+ stimulates PsaK insertion but is

not essential.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

13

PsaK insertion does not require the thylakoidal Sec machinery or Alb3

The data shown in Figs. 3 and 4 exclude the involvement of SRP or NTPs in PsaK insertion but it is

equally important to understand whether membrane-bound translocation machinery is involved. The

question of Alb3 involvement is critical because the homologous YidC protein is required for the

efficient insertion of every E. coli membrane protein tested to date [19], and the SecYEG complex

is also a prime candidate since this translocon is also used for some membrane proteins in bacteria

(reviewed in [1]). We addressed these possibilities in two ways. Previous studies [31] have shown

that the insertion of Lhcb1 is totally inhibited by pretreatment of the thylakoids with trypsin, and the

same study showed that translocation of Sec-dependent lumenal proteins is also completely

blocked. This technique provides a simple means of destroying both the membrane-bound Sec

apparatus and inhibiting integration by the SRP pathway. In previous studies using this approach we

have maintained a ∆µH+ by driving the ATP synthase in reverse in the dark (the synthase is highly

resistant to trypsin) and we used the same method in this study since insertion of PsaK is stimulated

by the ∆µH+.

Fig. 5 shows experiments in which thylakoids were treated with trypsin, washed with buffer

containing trypsin inhibitor to remove protease, and then assayed for their ability to import pre-23K

(a substrate for the twin-arginine translocation, or Tat pathway), Lhcb1 and PsaK. The pre-23K

imports serve as a test for the establishment of the ∆µH+ since transport of this protein into the

lumen is completely dependent on the proton gradient [1], and the data show that in the absence of

trypsin treatment this protein is indeed imported with high efficiency and processed to the mature

size, in total darkness. This observation confirms the presence of a ∆pH, and import is abolished by

trypsin treatment which has been shown previously to inactivate the Tat system [31]. Insertion of

Lhcb1 is also completely inhibited by this treatment; very little Lhcb1 is found associated with the

thylakoids after the incubation (lane T of the 'Trypsin' panel) and essentially no resistant

degradation product is found after protease-treatment (lane T+) and other experiments (not shown)

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

14

confirmed that import of a Sec substrate was also blocked. However, the upper panel shows that

PsaK still inserts into trypsin-treated thylakoids, which indicates that the Sec system is not required.

The specific question of Alb3 involvement was approached by more direct means. Pre-incubation of

thylakoids with polyclonal anti-Alb3 antibodies almost blocks Lhcb1 insertion without affecting the

Tat- or Sec-dependent pathways [13] and the same technique was used to test for its involvement in

PsaK insertion. Fig. 6A shows a control assay using Lhcb1, in which insertion was monitored after

incubation of the thylakoids with buffer (HM; as a control) with pre-immune antibodies (PI) or anti-

Alb3 antibodies. The pre-immune serum causes a slight inhibition of insertion (to 89% of the

control value) but the Alb3 antibodies reduce insertion efficiency to 27% of the control value. A

similar level of inhibition was observed previously [13]. In contrast, Fig. 6B shows that neither the

pre-immune nor the Alb3 antibodies affect insertion of PsaK into thylakoids, and the levels of urea-

resistant protein or TM1 or TM2 degradation products remain undiminished. These data clearly

indicate that PsaK is not dependent on Alb3 for insertion.

The C-terminal transmembrane span of PsaK can insert independently

The above data show that PsaK inserts by a relatively simple mechanism that does not rely on any

known translocation apparatus, including Alb3. This type of mechanism is highly unusual and we

have sought to obtain further details on the overall insertion mechanism. One possibility, proposed

for many membrane proteins [1], is that the two transmembrane spanning regions may form a

'helical hairpin' that is able to insert with high efficiency due to the simultaneous partitioning of two

hydrophobic regions. Several membrane proteins are known to form loop intermediates in which

the loop region is on the trans side of the membrane, and similar principles may operate for those

proteins, such as PsaK, where the loop remains on the cis side. We tested whether a single span of

PsaK can insert independently into the thylakoid membrane, by simply using the full precursor

protein instead of the mature PsaK construct, under conditions where cleavage by SPP is prevented.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

15

As explained above, the N-terminus of mature PsaK lies in the lumen, hence cleavage of the large

and highly charged presequence would appear to be essential before this N-terminal region can

translocate across the membrane. We therefore carried out thylakoid import assays under two

conditions: in the complete absence of stromal extract (using thylakoids that had been thoroughly

washed to remove residual SPP), and after protease-treating the thylakoids (to destroy any SPP on

the membrane surface). Mature-size PsaK was used as a control since this protein inserts under both

sets of conditions as shown above.

The data (Fig. 7) show that the mature-size PsaK ('PsaK' panel) behaves as in experiments shown

above and the TM1 and TM2 fragments again appear with a labeling ratio which was calculated to

be close to 3:1. Insertion occurs with both the washed and protease-treated thylakoids. However,

very different results are obtained when the full precursor protein is used ('pPsaK' panel). The upper

degradation fragment (TM2) is again observed after insertion into either washed or protease-treated

thylakoids but the lower band (TM1) is now completely absent. A low-intensity smear of label is

present beneath the TM2 band, presumably due to degradation of non-inserted PsaK regions, but no

band is present in the TM1 region. We conclude from this result that the N-terminal transmembrane

span is indeed unable to insert when the presequence is present, as predicted above, but the clear

presence of TM2 is strong evidence that this region is able to insert independently under these

conditions. These data also serve to reinforce the efficacy of the in vitro assay because they provide

a third line of evidence that the bands denoted TM1 and TM2 do indeed represent inserted

transmembrane regions; the N-terminal hydrophobic region is clearly highly susceptible to

proteolysis (or is simply removed when the thylakoids are washed after the insertion reaction) when

not inserted in the thylakoid membrane.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

16

DISCUSSION

Several thylakoid membrane proteins have been previously analyzed in terms of insertion

mechanism, and in this respect they fall into two broad categories. Lhcb1 follows a complex

pathway involving the input of numerous factors, both in the stroma and at the membrane surface,

while several signal peptide-bearing proteins use an apparently simpler insertion mechanism that

does not rely on any of the known protein machinery, although the issue of Alb3 involvement has

yet to be addressed. PsaK is unlike any of the above proteins in that both the N- and C-termini are

transported to the lumen, the protein is not synthesized with a signal-type peptide and it is not a

member of the LHC super-family of proteins.

The data from this study all point to a strictly SRP-independent insertion mechanism. Stromal

extract contains essentially all of the SRP but is not required at any stage, and insertion does not

depend at all on NTP hydrolysis. Both of these factors are critical for Lhcb1 insertion. FtsY

involvement can also be ruled out since this factor hydrolyzes GTP during its operating mechanism.

We can not rule out the possibility that other, as yet unidentified soluble factors may assist PsaK

insertion, and the slight inhibitory effect of apyrase does raise the possibility that proteins in the

wheatgerm translation system (eg chaperones) may aid insertion, but the data nevertheless indicate

that the insertion of PsaK is fundamentally different from that of Lhcb1. It is as yet unclear why

Lhcb1 is so dependent on SRP activity whereas other thylakoid membrane proteins studied to date

are not.

We also find no evidence for the involvement of membrane-bound translocation machinery in PsaK

insertion. Several studies have shown that trypsin treatment blocks the translocation of lumenal Sec

substrates, very strongly suggesting that the Sec apparatus is inactivated. This treatment slightly

inhibits PsaK insertion (as indeed it does for PsbY, [24]) but the effect is not marked and we

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

17

conclude that the Sec translocon does not play a major role in this pathway. We also find no

evidence for Alb3 involvement, since antibodies raised against Alb3 severely inhibit Lhcb1

insertion yet have no effect on the insertion of PsaK. On the basis of these data alone we can not

exclude the possibility that PsaK may interact with Alb3 in a manner which is not affected by the

antibodies used in this study. However, other ongoing studies in this lab (not shown) have

demonstrated that Alb3 is completely degraded when thylakoids are treated with trypsin under the

conditions used in this study (eg in Figs. 5 and 7) and, since this treatment does not affect PsaK

insertion at all, we conclude that Alb3 is not required for PsaK biogenesis. This finding is

significant because the homologous YidC protein plays a central role in the insertion of several

SRP/Sec-independent membrane proteins in E. coli [19]. This factor has come to be regarded as a

novel form of translocase in its own right, since the related Oxa1 protein also plays an important

role in membrane protein biogenesis in yeast [17,18] and there is no evidence as yet for additional

subunits in the mirochondrial Oxa1p complex. Our data thus indicate that PsaK is unique (to date)

because it is the only Alb3/YidC-independent membrane protein among those analyzed in bacteria

and plant thylakoids

In general, the topology adopted by PsaK is consistent with the ‘positive-inside’ rule [30] which

states that positively-charged residues are found more frequently on the cis side of the membrane.

The stroma-exposed loop region contains 4 basic residues whereas the C-terminal lumenal tail

contains only one and the N-terminal region does not contain any [24]. However, it is presently

unclear why insertion is stimulated to some extent by the thylakoidal ∆pH. In bacteria and

mitochondria, the insertion of many membrane proteins is stimulated by the proton motive force

[1,17,18] and the same applies to the Alb3-dependent insertion of Lhcb1 in thylakoids [9,11,12].

However, these effects probably reflect the harnessing of the ∆µH+ by the translocation machinery

(the Sec and/or Oxa1-type apparatus) and these factors are not required for PsaK insertion. Possibly,

there is a mildly stimulatory electrophoretic effect on the translocation of the C-terminal region of

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

18

PsaK, which contains a single acidic residue. However, this point remains to be investigated in

detail.

The Sec/Alb3-independent nature of the insertion mechanism raises the strong possibility that

insertion of PsaK occurs spontaneously upon reaching the thylakoid membrane, and such

mechanisms have been postulated before on theoretical grounds and following studies on

membrane-interactive peptides and toxins (reviewed in [32]). However, further work is required to

address this possibility, and one argument against this possibility is that PsaK may then be able to

interact with other membranes (eg the envelope) in the absence of a specific and dedicated targeting

system. Possibly, PsaK is predisposed to insert only into thylakoid-type lipids, and the thylakoid

membrane is indeed very unusual in terms of lipid composition, being composed primarily of

galactolipids which are chemically very different to phospholipids (reviewed in [33]). Further work

is certainly required to determine whether such a lipid-based sorting process operates for PsaK

insertion, or whether novel forms of translocation apparatus are involved.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

19

Acknowledgements

This work was supported by an Engineering and Physical Sciences Research Council studentship to

C.W. and EPSRC Biosciences Interface Network grant GR/M91105 to A.R. and C.R., and by

Biotechnology and Biological Sciences Research Council grant C07900 to C.R. and by National

Science Foundation grant MCB-9807826 to R. H.

Abbreviations

AMP-PNP (5’adenylylimidodiphosphate); GMP-PNP (5’guanylylimidodiphosphate); LHC protein

(light-harvesting chlorophyll a/b-binding protein); SDS-PAGE (SDS polyacrylamide gel

electrophoresis); Tricine (N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine).

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

20

REFERENCES

[1]. Dalbey, R.E. and Kuhn, A. (2000). Annu Rev Cell Dev Biol. 16, 51-87.

[2]. De Gier, J-W. L., Mansournia, P., Valent, Q.A., Phillips, G.J., Luirink, J. and von Heijne, G.

(1997). FEBS Lett. 399, 307-309.

[3]. Gebert, J.F., Overhoff, B., Manson, M. and Boos, W. (1988). J. Biol. Chem. 263, 16652-16660.

[4]. Traxler, B. and Murphy, C. (1996). J. Biol. Chem. 271, 12394-12400.

[5]. MacFarlane, J. and Müller, M. (1995). Eur. J. Biochem. 233, 766-771.

[6]. Ulbrandt, N.D., Newitt, J.A. and Bernstein, H.D. (1997). Cell 88, 187-196.

[7]. Valent, Q.A., de Gier, J-W. L., von Heijne, G., Kendall, D.A., ten Hagen-Jongman, C.M.,

Oudega, B. and Luirink, J. (1997). Mol. Microbiol. 25, 53-64.

[8]. Luirink, J., ten Hagen-Jongman, C.M., van der Weijden, C.C., Oudega, B., High, S.,

Dobberstein, B. and Kusters, R. (1994). EMBO J. 13, 2289-2296

[9]. Li, X., Henry, R., Yuan, J., Cline, K. and Hoffman, N.E. (1995). Proc. Natl. Acad. Sci. USA 92,

3789-3793.

[10]. Kogata, N., Nishio, K., Hirohashi, T., Kikuchi, S. and Nakai, M. (1999). FEBS Lett. 329, 329-

333.

[11]. Tu, C.J., Schuenemann, D. and Hoffman, N.E. (1999) J. Biol. Chem. 274, 27219-24.

[12]. Mori, H., Summer, E.J., Ma, X. and Cline, K. (1999). J. Cell Biol. 146, 45-55.

[13]. Moore, M., Harrison, M.S., Peterson, E.C. and Henry, R. (2000). J. Biol. Chem. 275, 1529-

1532.

[14]. Schuenemann, D., Gupta, S., Persello-Cartieaux, F., Klimyuk, V.I., Jones, J.D.G., Nussaume,

L. and Hoffman, N.E. (1998). Proc Natl Acad Sci U S A. 95, 10312-10316.

[15]. DeLille, J., Peterson, E.C., Johnson, T., Moore, M., Kight, A. and Henry, R. (2000). Proc.

Natl. Acad. Sci. U S A. 97, 1926-1931.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

21

[16]. Tu, C.J., Peterson, E.C., Henry, R. and Hoffman, N.E. (2000). J. Biol. Chem. 275, 13187-

13190.

[17]. Herrmann, J.M., Neupert, W. and Stuart, R.A. (1997). EMBO J. 16, 2217-2226.

[18]. Hell, K., Neupert, W. and Stuart, R.A. (2001). EMBO J. 20, 1281-1288.

[19]. Samuelson, J. C., Chen, M., Jiang, F., Moeller, I., Wiedmann, M., Kuhn, A., Phillips, G.J. and

Dalbey, R.E. (2000). Nature 406, 637-641.

[20]. Kuhn, A. (1995). FEMS Micro. Rev. 17, 185-190.

[21]. Scotti, P.A., Urbanus, M.L., Brunner, J., de Gier, J.W., von Heijne, G., van der Does, C.,

Driessen, A.J.M., Oudega, B. and Luirink J. (2000). EMBO J. 19, 542-549.

[22]. Michl, D., Robinson, C., Shackleton, J.B., Herrmann, R.G. and Klösgen, R.B. (1994). EMBO

J. 13, 1310-1317.

[23]. Kim, S.J., Robinson, C. and Mant, A. (1998). FEBS Letts. 424, 105-108.

[24]. Kjærulff, S., Andersen, B., Nielsen, V. S., Møller, B. L., and Okkels, J. S. (1993) J. Biol.

Chem. 268, 18912-18916.

[25]. Adam, Z. and Hoffman, N.E. (1993). Plant Physiol. 102, 35-43.

[26] James, H.E., Bartling, D., Musgrove, J.E., Kirwin, P.M., Herrmann, R.G. and Robinson, C.

(1989). J. Biol. Chem. 264, 19573-19576.

[27]. Thompson, S. J., Robinson, C. and Mant, A. (1999) J. Biol. Chem. 274, 4059-4066.

[28]. Breyton, C., de Vitry, C. and Popot, J.-L. (1994) J. Biol. Chem. 269, 7597-7602.

[29] Jensen, P.E., Gilpin, M., Knoetzel, J. and Scheller, H.V. (2000). J. Biol. Chem. 275, 24701-

24708.

[30] Gavel, Y., Steppuhn, J., Herrmann, R.G. and von Heijne, G. (1991). FEBS Lett. 282, 41-46.

[31]. Robinson, D., Karnauchov, I., Herrmann, R.G., Klösgen, R.B. and Robinson, C. (1996). Plant

J. 10, 149-155.

[32]. White, S.H. and Wimley, W.C. (1999). Annu. Rev. Biophys. Biomol. Struc. 28, 319-365.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

22

[33]. Douce, R. and Joyard, J. (1996). In D.R. Ort, C.F Yocum, eds. Advances in Photosynthesis,

Vol 4: Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht,

The Netherlands, pp69-101

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

23

Figure Legends

Figure 1 Schematic diagram of the ‘horse-shoe’ topology adopted by PsaK.

The protein forms two transmembrane alpha helices, with both N- and C-termini in the thylakoid

lumen. The positively-charged loop region on the stromal side of the membrane is accessible to the

protease trypsin, yielding two degradation products corresponding to the bulk of the two

transmembrane helices and their small lumenal tails. Recent crystallographic data indicate that one

of the transmembrane spans of cyanobacterial PsaK crosses the membrane at a tilted angle (N.

Krauss, personal communication), and therefore may form a more open ‘horse-shoe’ than

represented here. The Figure also shows the complete amino acid sequence of pPsaK with the

transit peptide (T. peptide) given on the upper line and the transmembrane domains underlined and

methionine residues shown in bold. Potential trypsin-sensitive basic residues in the stromal loop

region are shown in bold italics. The sequence of the mature PsaK construct is also indicated (with

the introduced initiation methionine shown in parentheses).

Figure 2. Barley pPsaK is efficiently imported by intact pea chloroplasts.

12.5 µl in vitro-translated pPsaK (lane Tr) was incubated with intact pea chloroplasts equivalent to

50 µg chlorophyll for 20 min in the light. After the incubation, chloroplasts were washed, reisolated

and fractionated, then analyzed by SDS-PAGE and fluorography. Lane C, total washed

chloroplasts; lane C+, thermolysin-treated chloroplasts; lane S, stromal extract; lane T, thylakoid

membranes; lane Turea, thylakoid membranes subjected to severe washing with 6.8 M urea/20 mM

Tricine-NaOH, pH 8; lane 1, trypsin-digestion of the thylakoid membranes from lane T; lane 2, the

proteolysed membranes from lane 1 subjected to urea-extraction; lane 3, the membranes from lane

Turea digested with trypsin; pPsaK, the precursor of PsaK, PsaK, the mature protein; TM1 and

TM2, the degradation products corresponding to transmembrane spans 1 and 2 of the mature protein

respectively.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

24

Figure 3 Insertion of pPsaK and PsaK into isolated pea thylakoids.

A: 5 µl in vitro-translated pPsaK (lane Tr) was incubated with isolated pea thylakoids equivalent to

20 µg chlorophyll for 20 min in the light. After the incubation, the thylakoids were washed with

HM and TB (see Experimental Procedures) and then urea-extracted, before digestion with trypsin

(lane T+). Samples were analysed by SDS-PAGE and fluorography.

B: 5 µl in vitro-translated PsaK (lane Tr) was incubated with isolated pea thylakoids (20 µg

chlorophyll) in the presence of stromal extract (SE), in the absence of stromal extract (HM), in the

absence (-) or presence (+) of 2 U apyrase, and in the presence of 0.5 mM AMP-PNP or 0.5 mM

GMP-PNP (as indicated above the lanes). After a 20 minute incubation in the light, the membranes

were washed with HM and TB, before being subjected to extraction with urea (lower panel, +Urea),

the urea-resistant protein being denoted as PsaK. Next the urea-washed membranes were digested

with trypsin (upper panel, + Trypsin), where the characteristic degradation products are denoted by

TM1 and TM2. An aliquot of the translation mixture alone was also digested with trypsin (lane

Tr+). The samples were analysed by SDS-PAGE and fluorography. Insertion efficiencies (beneath

the lower panel, quoted as %), relative to the HM buffer control (100%) were calculated by using a

phosphorimager to measure the densities of the urea-resistant mature PsaK bands.

C: As a control, pea pLhcb1 (lane Tr) was translated in vitro and incubated with isolated pea

thylakoids, in the absence (HM) or presence (SE) of stromal extract, and in the absence (-) or

presence (+) of 2 U apyrase, for 20 min in the light. After the incubation, the thylakoids were

washed with HM and TB, before being subjected to both urea-extraction and digestion by trypsin,

which yielded the normal degradation product (marked DP) when correct insertion occurred. An

aliquot of the translation mixture alone was also digested with trypsin (lane Tr+).

Figure 4. Insertion of PsaK is stimulated by the thylakoidal ∆µH+.

4A. Intact pea chloroplasts equivalent to 50 µg chlorophyll were incubated with 12.5 µl in vitro-

translated pPsaK (lane Tr) in the absence (Control) and presence (+ Nigericin) of the proton

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

25

ionophore nigericin (at 2 µM final concentration), for 20 min in the light. After the import

incubation, the chloroplasts were washed, fractionated and analysed by SDS-PAGE and

fluorography. Lanes C, total washed chloroplasts; lanes C+, thermolysin-treated chloroplasts; lanes

S, stromal extract (prepared, as always, in the presence of 10 mM EDTA, to prevent residual

thermolysin activity from degrading any stromal intermediates); lanes T, thylakoid membranes;

lanes T+, trypsin-treated thylakoid membranes; pPsaK, precursor of PsaK; iPsaK, an intermediate

form of PsaK; PsaK, mature protein; TM1 and TM2, degradation products corresponding to

transmembrane spans 1 and 2 respectively.

4B.Isolated pea thylakoids equivalent to 20 µg chlorophyll were incubated with 5 µl in vitro-

translated PsaK (lane Tr), in the presence (SE) and absence (HM) of stromal extract, and in the

presence (N) and absence (C) of 2 µM proton ionophore nigericin, for 20 min in the light. After the

insertion incubation, the membranes were washed with HM and TB, before being extracted with

urea (upper panel, +Urea). After urea-extraction, the membranes were digested with trypsin (lower

panel, + Trypsin). Urea-resistant mature protein is marked PsaK, while the protease degradation

products corresponding to transmembrane spans 1 and 2 are marked TM1 and TM2 respectively.

After the experiment the samples were analysed by SDS-PAGE, and insertion efficiencies (shown

below the lower panel, in %) measured by a phosphorimager to calculate the amounts of urea-

resistant mature PsaK relative to the control sample (HM, C).

Figure 5. Pre-digestion of the thylakoid membranes with trypsin does not prevent insertion of PsaK.

Thylakoid membranes were digested with 60 µg/ml trypsin (Trypsin) or buffer (Control) and the

chloroplast ATPase was subsequently activated to generate a ∆pH in the dark, as described in detail

in reference 31. The activated membranes (20 µg chlorophyll) were incubated with 5 µl in vitro-

translated PsaK, pLhcb1 or p23K (lanes Tr) in the dark for 30 min, with all manipulations being

carried out under a dim, green, safe light. After the incubation, the membranes were washed with

HM and reisolated. PsaK and Lhcb1 samples were washed further with TB, before being subjected

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

26

to urea-extraction (lanes T) and then trypsin-digestion (lanes T+). 23K samples were analysed

directly after washing with HM (lanes T) or after digestion with 0.2 mg/ml thermolysin for 40 min

on ice (lanes T+). All samples were analysed by SDS-PAGE and fluorography. The insertion

efficiencies of PsaK (values in %, relative to the control sample) were measured using a

phosphorimager. PsaK, mature protein; TM1 and TM2, trypsin degradation fragments

corresponding to transmembrane helices 1 and 2 of PsaK; pLhcb1, precursor of Lhcb1; DP, trypsin

degradation fragment of inserted Lhcb1; p23K, precursor of 23K; 23K, mature protein.

Figure 6. Anti-Alb3 antibodies inhibit the insertion of Lhcb1 but not PsaK. Isolated pea thylakoids

were pre-incubated with anti-Alb3 antibodies (Alb3), pre-immune serum (PI) or import buffer

(HM) for 2h on ice, as detailed in reference 15. After this period the thylakoids were incubated with

pLhcb1 or PsaK as indicated and analyzed by protease treatment as described in Fig. 3.

Figure 7. Independent insertion of the C-terminal transmembrane span of pPsaK. The full precursor

of PsaK (pPsaK) or the mature-size construct (PsaK) were incubated either with thylakoids that had

been washed 3 times in HM buffer to remove stromal extract (denoted as 'Thyl') or with thylakoids

that had been treated with 0.15 mg/ml proteinase K for 30 min on ice and then washed 3 times with

HM buffer (denoted as 'PK-Thyl'). After incubation, samples of the thylakoids were analysed

directly (-) or after trypsin treatment as in previous Figures (+). Lanes Tr: translation products.

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

27

Figure 1.

Stroma

Thylakoid membrane

Thylakoidlumen

+ Trypsin 0.2 mg/ml

N C NC

TM1 TM1TM2 TM2

Radiolabeled methionine residue

TM Transmembrane span

++

+ -

-

-

+

T. peptide: MASQLSAMTSVPQFHGLRTYSSPRSMATLPSLRRRRSQGIRCPsaK: (M)DYIGSSTNLIMVTTTTLMLFAGRFGLAPSANRKATAGLKLEA RESGLQTGDPAGFTLADTLACGAVGHIMGVGIVLGLKNTGVLDQIIG

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

28

Figure 2

- 6.2

- 8.2

- 16.9- 14.4

- 10.8

Tr C C+ S T Turea 1 2 3

pPsaK -

PsaK -

TM2TM1

kDa

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

29

Figure 3

Tr T T+

Tr Tr+ - + - +

SE HM GMP-PNP AMP-PNP------------ ------------ ------------- ------------

SE HM SE HM

- TM2- TM1

pPsaK -

PsaK -

PsaK -

+ Trypsin

+ Urea

A B

Efficiency: 83 41 100 71 117 109 93 85

pLhcb1 -

- DP

- + - +Tr Tr+-------- --------- HM SEC

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

30

Figure 4A

Tr C C+ S T T+ C C+ S T T+------------------------------- -----------------------------

Control + Nigericin

- pPsaK

- PsaK

- TM2- TM1

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

31

Figure 4B

Tr N C N C------------- -------------

SE HM

- PsaK

- TM2- TM1

+ Urea

+ Trypsin

Efficiency: 42 55 75 100

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

32

Figure 5

pLhcb1 -

p23K -

- 23K

- DP

PsaK -- TM2- TM1

Tr T T+ T T+ --------- -------- Control Trypsin

Efficiency: 100 70

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

33

Figure 6

Tr. HM PI Alb3 HM PI Alb3------------------ ---------------------Washed Prot. Treated

100% 89% 11%

- DP

Tr. HM PI Alb3 HM PI Alb3------------------ ---------------------Washed Prot. Treated

100% 93% 109%

- TM2- TM1

pLhcb1 -

PsaK -

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

34

Figure 7

- PsaK

- TM2- TM1

pPsaK -

Tr - + - + Tr - + - +

--------------------- ----------------------pPsaK PsaK

Thyl: + + + +PK-thyl: + + + +

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from

Alexandra Mant, Cheryl A. Woolhead, Misty Moore, Ralph Henry and Colin Robinsonfunctional Albino3

occurs in the absence of signal recognition particle, nucleoside triphosphates or Insertion of PsaK into the thylakoid membrane in a 'horse-shoe' conformation

published online July 12, 2001J. Biol. Chem. 

  10.1074/jbc.M102914200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on February 6, 2018http://w

ww

.jbc.org/D

ownloaded from