Synthesis of Several Light-Harvesting Complex I Polypeptides Is Blocked by Cycloheximide in Symbiotic Chloroplasts in the Sea Slug, Elysia chlorotica (Gould): A Case for Horizontal Gene Transfer Between Alga and Animal?

11 Pages·2001·4.8 MB·

by  Jeffrey J Hanten

Reference: Bio/. Bull. 201: 34-44. (August 2001)
Synthesis of Several Light-Harvesting Complex I
Polypeptides Is Blocked by Cycloheximide in
Symbiotic Chloroplasts in the Sea Slug, Elysia
chlorotica (Gould): A Case for Horizontal Gene
Transfer Between Alga and Animal?
JEFFREY J. HANTEN1'2 AND SIDNEY K. PIERCE2 *
1 Department ofBiology. University ofMan-land, College Park, Maryland 20742; and
2 Department ofBiology, University ofSouth Florida. Tampa, Florida 3362G
Abstract. The chloroplast symbiosis between the asco- oles inside the animal's cells. Rarer, but not uncommon, are
glossan (=Sacoglossa) sea slug Elysia chlorotica and plas- intracellular symbioses occurring with intact algal chloro-
tids from the chromophytic alga Vaitcheria litorea is the plasts that are captured by specialized cells within the
longest-lived relationship of its kind known, lasting up to 9 animal. In particular, several species of ascoglossan
=
months. During this time, the plastids continue to photosyn- ( Sacoglossa) (Opistobranchia) sea slugs capture intact,
thesize in the absence ofthe algal nucleus at rates sufficient functional plastids from their algal food source and retain
to meet the nutritional needs of the slugs. We have previ- them within specialized cells lining the mollusc's digestive
ously demonstrated that the synthesis of photosynthetic diverticula. This phenomenon has been termed chloroplast
proteins occurs while the plastids reside within the diver- symbiosis (Taylor. 1970) or kleptoplasty (Clark et al..
ticular cells ofthe slug. Here, we have identified several of 1990). The sequesteredplastids continue to photosynthesize
these synthesized proteins as belonging to the nuclear- for periods ranging from a few days to a few months,
encoded family of polypeptides known as light-harvesting depending on the species (Greene, 1970: Hinde and Smith,
complex I (LHCI). The synthesis ofLHCI is blocked by the 1974; Graves et al., 1979; Clark et al., 1990).
cytosolic ribosomal inhibitor cycloheximide and proceeds The longest such association, lasting as long as 9 months,
in the presence of chloramphenicol, a plastid ribosome is found in Elysia chlorotica (Gould), which obtains sym-
itnhheibniutcolr,eairndDicNatAinogfthtahtetshleugg.enTeheesnecordeisnulgtsLHsCugIgersetsitdheastina brieoatic(C.plaAsgtiadrsdhf)rom(Wetshet,ch1r9o7m9o;phPyiteirccealegtaalV.a.uc1h9e9r6i).a lTihtoe-
horizontal transfer of the LHCI gene from the alga to the association begins at metamorphosis ofthe slug from plank-
slug has taken place. tonic veliger tojuvenile. In laboratory cultures, filaments of
V. litorea must be present for metamorphosis to take place
Introduction
(West et al.. 1984). Veligers home in, attach to the fila-
Most alga-animal symbioses are extracellular associa- ments, and metamorphose intojuvenile slugs over the next
tions between two genetically distinct organisms. The alga 24 h. The juveniles eat the algal filaments and sequesterthe
is usually located extracellularly or enclosed within vacu- chloroplasts within one of at least two morphologically
distinct types of epithelial cells lining the walls of the
Received 22 September 2000; accepted 19 April 2001. digestive diverticula (West et al., 1984). Once the plastids
* To whom correspondence should be addressed. E-mail: are sequestered, the slugs can sustain photosynthesis at rates
[email protected] sufficient to satisfy the nutritional needs for the complete
fucAobxbarnetvhiianticohnlso:roCphAyPl,l aclhelobrianmdpihnegnipcrootle:insC;HLXH.C.cylcilgohhte-xhairmviedset;ingFCcPoCm,- life cycle of the slug, when provided with direct light and
plex; PSI; photosystem I. carbon dioxide (Mujer et al., 1996; Pierce et al., 1996).
34
SYMBIOTIC PLASTID GENES IN SLUGS 35
Even in nature the slugs obtain most of their energy from aerated, artificial seawater (ASW: Instant Ocean. 925-1000
photosynthesis (West. 1979). mosm) on a 16/8-h light/dark cycle (GE cool-white fluores-
The longevity ofthis relationship in E. chlorotica makes cent tubes. 15 W).
it especially interesting. Photosynthesis requires the contin- Sterile cultures of Vaucheria litorea were maintained in
uous synthesis of a variety of chloroplast proteins because enriched ASW (400 mosm) [modified from the F/2 medium
many ofthem, including those used in light harvesting, are (Bidwell and Spotte. 1985)]. The alga was grown at 20 C
rapidly degraded and must be replaced (Greenberg et cil.. on a 16/8-h light/dark cycle (GE cool-white fluorescent
1989; Mattoo et ai, 1989; Barber and Andersson, 1992; tubes; 40 W). and the medium was changed weekly.
Wollman et ai. 1999). Furthermore, photosynthesis re-
quires the interaction of as many as 1000 proteins, only Inhibitor treatments andplastidprotein labeling
about 13% ofwhich are coded in the plastid genome (Mar-
tin and Herrmann, 1998). In the plant cell, substantial nu- All reagents used were molecular bio-grade (DNase-,
RNase-. and protease-free) purchased from Sigma unless
clear input is required to sustain photosynthetic function, in otherwise noted. Effective concentrations ofCHX and CAP
theform ofdirect coding ofthe proteinsas well asproviding
rCit(sneheoBeqvneuemsrirmiorradseeyletda-r,npLihskointolwgetiefpsootstnroayhopnnettltdhahselsueietSrrtvicpiechrlciminisstpolidrrfunatoggc.tesnetluihl1cinau9sltsl9eat1rarah;erreletaWraltaoosinnvnyldsegnlpletoymevhrxiaetttssnrhyiaaoz-roneeptftddl.atushriiHtene,oigpdwutle1hla9eiavs9ntte9piisr)uode,.tn-s cei(wmntui1ehhrt6riavo0ebneciosh/tlood,)(gpnePrdtmwioreealtirresamcilienu,nseesrsteyditdnaobticoteh,ksomeospc1miiio9nesrn9hsici6.ocbe)ani.ntltTlCSrtwyaOrHtoSaiXnwoscintlyt(oath25toi0sfmoooingmnluiirgotcminalmlsr"l7ilu0b"d1goS)so1s,swopieamln-tsaerosastetbua;sislsdpoeColdwanAuensttPtdeeo
/qiaunecnsldtudeoirtnehgdertpshlea(slPtaiiredgrsecesoufebtuEan.ii,tcho1lf9o9rR6ou;tBiMicuasjCe(OrP,ieeDtrlcae,i,De2t1,9a9iC6,P;431G9,r9e6ce)yn,t viwineahiligsbhittcoorna,tbaoaiunntdin1ig.n2cA5ubSga,Wtewd(e1ru0en0d0pelramcoiensdtmen)isnetaonldigglhattshse(1sa5cpi0pnrtWoilp,lraitGaitoEen
Cool Beam incandescent indoor flood lamp) at 20 C in a
eiPtdieeanritc,ief2i0ee0dt0)ta.oi,Adlat1te9h9oa6ur;gehGparllaelseotnifdtehetencasiyo,ndteh2de0s0(i0Mz)ue.jdeptrlwaoesttigadriop,ruop1ts9e9i6no;sf gmeentthliyonaignietat(i0n.g7wMatBeqr bmalth".1,Afttrearns-1[3h5.S]2-0me/tihCiiomniln"e,1 [I35CSN])-
synthesized plastid proteins can be distinguished pharma- was added, and the slugs were incubated for an additional
6 h. previously demonstrated to provide ample time to
cologically: those inhibited by cycloheximide (CHX). an
SOS cytosolic ribosome inhibitor (Obrig et ai, 1971). and incorporate radioactive label intothe plastidproteins (Pierce
tphrootseeininhsiybnittheedsibsyocnhl7o0rSampplhaesntiicdolan(dCAmPi)t,ochwohnidcrhiailnhriibbiot-s eetthaai,nol19/9A6S)W. (Avd/dvi)tisoonlaultiosnlupglsCusAwPe[3r5eS]i-nmceutbhaitoendinien t0o.s0e2r5v%e
as a control for the carrier in treatments.
somes (Lamb et ai, 1968: Stone and Wilke. 1975).
Because the inhibition by CHX suggests that the genes
for several plastid proteins must reside in the nuclear DNA, Chloroplast isolation andprotein separation
we have done some experiments to identify these proteins Chloroplasts were isolated from slugs by using a centrif-
and test that possibility. Our present study reports the iden- ugation protocol. The slugs were homogenized in the pres-
tification of several of the CHX-blocked proteins as mem- ence of the mucolytic agent N-acetyl-cysteine (500 mM).
bers of the light-harvesting complex 1 (LHCI). a family of and the homogenate was filtered successively through
pigment-binding proteins responsible for collecting radia- cheesecloth, Miracloth (Calbiochem), and then nylon mesh
tion energy from sunlight and transferring it to photosystem (60 ;um to 10 jitm) to remove large debris and the copious
I (PSI). LHCI proteins are encoded by the Lhca genes in the amount of mucus the animals produce. The plastids were
nuclear genome ofall the plants and algae whose genomes purified on apre-formed. 25% Percoll (v/v) gradient, which
havebeen examined (Jansson, 1994. 1999; Green and Durn- provides a very pure fraction containing large numbers of
ford, 1996; Durnford et ai, 1999; Wollman et ai, 1999). intact plastids (Pierce et ai, 1996). In this experiment, the
This result suggests that the LHCI genes have been some- lowest green band containing labeled plastids was isolated
how transferred from the algal nucleus to the slug's DNA. from the gradient by using a flamed Pasteur pipette, and
residual Percoll was removed by centrifugation. The puri-
Materials and Methods fied chloroplast pellets were resuspended, lysed by freeze-
Animals and alga thawing. and stored at 20C until use. The incorporation
ofradioactive label was determined by a liquid scintillation
Specimens ofElysici chlorotica were collected in both the counter (Beckman LS60001C). and the protein content was
spring and fall from an intertidal marsh near Menemsha determined using the modified Lowry assay (Peterson,
Pond on the island of Martha's Vineyard, Massachusetts. 1977). The resulting specific activity was calculated as
The slugs were maintained in 10-gallon aquaria at 10 C in counts per minute (cpm) (jug protein)^'. Chlorophyll con-
36 J. J. HANTEN AND S. K. PIERCE
tent was determined by extracting the pigment in 80% identify the coincidence ofantibody binding and radioactive
acetone, then measuring the extract absorbance spectropho- incorporation in the presence of each inhibitor.
tometrically at 652 nm. The results were calculated as As a control to confirm that the CAP was blocking
micrograms per microliter according to standard equations plastid-directed protein synthesis and that CHX was not,
(Joyard et al., 1987). parallel measurements were run to monitor cytochrome /
Sodium dodecyl sulfate-polyacrylamide gel electro- (cyt/) synthesis. Earlierexperiments conducted on E. clilo-
phoresis (SDS-PAGE) autoradiography was used to assess rotica have demonstrated that cyt / is synthesized in the
the effects of CHX and CAP on the pattern of protein slugs and is encoded in the plastid DNA (Green et al.,
synthesis. The plastid lysates obtained from the above pro- 2000). Thus, if CHX and CAP are working as expected,
cedure were boiled for 2 min in Tris-HCl (pH 6.8)-10% their effect on cyt / and any nuclear-encoded proteins
SDS (w/v) buffer containing 5% /3-mercaptoethanol (|3- should be opposite. Anti-cyt/, raised to P. cmentum cyt/,
ME) (v/v). The solubilized proteins were loaded in equal was also a gift of Professor Gantt.
amounts onto 15% SDS-polyacrylamide gels and separated
by electrophoresis (Laemlli, 1970). The gels were stained
Immunoprecipitations
with Coomassie brilliant blue, dried, and exposed to film
(Kodak Biomax MR) for 2 to 30 days at -80C, depending Immunoprecipitations were conducted to confirm the
on the level of radioactive label present. Approximate mo- identity of the radioactive immunolabeled bands on the
lecular masses ofthe proteins were determined by compar- western blots, using a modified version of the protocol
ison to the migration distances of known molecular weight previously used to precipitate proteins from isolated E.
standards (BioRad, broad-range kaleidoscope) run in adja- chlorotica plastids (Pierce et al., 1996). Plastid proteins
mM mM
cent lanes on each gel. were solubilimzedMin lysing bumffMer ( 10 Tris-HCl, 10
EDTA, 150 NaCl, 1 PMSF, 1% (v/v) Nonidet
P-40, pH 8.0), using equal amounts of chlorophyll per
Immunoblot identification ofplastidproteins sample, mixed with a small amount ofProtein-A Sepharose
beads toeliminate nonspecific binding, and incubated on ice
After the plastid isolation and protein separation via with occasional agitation. The beads were removed by cen-
SDS-PAGE as described above, the proteins were electro- trifugation and discarded, the supernatant was saved, and
phoretically transferred (30 V, 4 C, overnight) to PVDF the appropriate antibody was added to the lysate and rotated
membranes (Immobilon-P; Millipore) (Towbin et til.. overnight (4 C). Protein-A beads, swelled in washing
mM mM mM
1979). As additional controls, V. litorea chloroplasts [iso- buffer (50 Tris-HCl, 5 EDTA, 150 NaCl, 1
mM
lated and purified using a 30% to 75% Percoll step gradient PMSF, 0.1% (v/v) Nonidet P-40, pH 8.0), were added
as previously described (Pierce etal., 1996)] and thylakoids the following morning and rotated (3 h, room temperature).
from the red alga Porphyridium cmentum (generously do- The antigen-antibody-protein-A Sepharose bead com-
nated by Professor Elisabeth Gantt, University of Mary- plexes were washed several times in washing buffer and
land), were lysed. and the proteins were separated electro- removed by centrifugation. In the case ofcyt/, the antigen-
phoretically and transferred to membranes as above. The antibody-protein-A Sepharose bead complexes were resus-
membranes were blocked with 5% (w/v) dehydrated milk pended in 10.0Murea. 10% SDS (w/v), 5% |3-ME (v/v), pH
dissolved in Tris-buffered saline (TBS) (Tris-base 50 mM. 12.5, and boiled for 10 min to liberate the cyt/antigen. The
NaCl 0.9%, pH 7.5) for 1 h at room temperature, washed solution was centrifuged, the supernatant was removed, and
twice in TBS for 10 min, and treated with primary antibody the beads were discarded. The supernatant proteins were
for 1 h. In this case, the primary antibody was a polyclonal separated by SDS-PAGE as described above, and the gel
antibody to LHCI which was produced in a rabbit using a was autoradiographed.
22-kDa, recombinant LHCI polypeptide produced from a The LHCI antibody-antigen complex could not be broken
clone ofthe LhcaRI gene ofP. cmentum (Grabowski et al., efficiently with any treatment, which prevented the visual-
2000) (also provided by Professor Gantt) ["/?/" indicating it ization of the labeled LHCI proteins via SDS-PAGE. Al-
is a rhodophyte gene (Tan et al., 1997a)] as the antigen though this was unexpected, it is not unusual and may have
combined with Freund's adjuvant in a standard immuniza- been caused by a numberoffactors. The presence ofseveral
tion procedure. After binding of the primary antibody, the different LHCI polypeptides with varying isoelectric points,
membranes were washedtwice as above and incubated with ranging between 4.5 and 9.5 (De Martino et al., 2000).
secondary antibody, anti-rabbitconjugated hydrogen perox- makes it very difficult to create optimal reaction conditions
idase, for 1 h. Afterwashing, the bands were visualized with for each one. The polyclonal antibody molecules bind to all
a 4-chloro-l-napthol and hydrogen peroxide reaction ac- the LHCI polypeptides as well as to each other, creating a
cording to manufacturer's instructions. The immunolabeled large antigen-antibody complex with a core inaccessible to
western blots were exposed to film as described above to the chemicals necessary to liberate the antigen. Very few
SYMBIOTIC PLASTID GENES IN SLUGS 37
researchers have attempted LHC immunoprecipitations be- (kDa) CON CHX CAP
cause of the pitfalls involved in precipitating inner-mem- 218_l
brane proteins (Anderson and Blobel, 1983). Instead, other
protocols have been designed using mild detergents to ex-
tract intact photosystem holocomplexes from the thyla-
koids. followed by protein separation on sucrose density
gradients (Fawley and Grossman, 1986; Buchel and Wil-
helm, 1993: Wolfe end., 1994; Schmid etid.. 1997). These
isolations require large amounts of starting material
(Schmidetid.. 1997) that greatlyexceedwhat is available to 43.5
us in the slugs. So, instead, we used the LHCI antibody to
demonstrate that LHCI had incorporated radioactivity.
Following the procedure described above, the protein A 33.9_
Sepharose beads were reacted with anti-LHCI and then with
a radiolabeled plastid protein extract. The antigen-anti-
body-protein-A Sepharose bead complexes were repeatedly
washed by centrifugation until the radioactivity in the su-
pernatant was reduced to background. The washed antigen-
antibody-protein-A Sepharose bead complexes were resus-
pended in optifluor (Packard), and radioactivity was
17.4_
determined by a scintillation counter. Controls for nonspe-
cific binding to protein-A Sepharose beads were conducted
with the same procedure, but without the addition of the
LHCI antibody. Counts per minute resulting from nonspe-
cific binding were subtracted from experimental values for
7.6_
each inhibitortreatmentandcontrols, andthe final datawere
converted to cpm (jug chlorophyll)"' (ju,g protein)"1. The
normalized data were averaged and expressed in terms of
percent of control for each inhibitor.
Results
Plastidprotein synthesis and identification
Figure 1. Coomassie brilliant blue-stained 15% SDS-PAGE gel of
The Coomassie-stained SDS-PAGE gels of protein ex- proteins extracted from isolated Elysia chloroplasts. The protein bands
rtreagcatrsdlfersosmofisotlhaeteidnhsilbuigtoprlapsrteisdesntw,ereeithseirmiClaHrXtoocronCtAroPl,s (viCsOuaNlirzeefderasretoidceonnttircoall).reAgparpdrloexsismoaftethemoilnehcibuiltaorrwtreeiagthmtesnta,reCHinXdicoarteCdAtPo
the left.
indicating no difference in the protein composition of the
plastids after treatment (Fig. 1). However, autoradiograms
of SDS-PAGE gels ofplastid proteins extracted from slugs identify a band with a molecular weight corresponding to
vienrcyubdaitfefdereinnttphaettperrenssenocfeproofte[i3n5Ss]y-nmtehtehsiisonoicnceurinidnitchaetesltuhgast csyatm/e, gceolnsfisrhmoiwng[1i5tsS]i-demnettihtiyo(nFiinge. 4i)n.coAruptoorraatdioinogirnatomscyotf/thien
between controls and inhibitors as well as between inhibi- the presence ofCHX. but not in the presence ofCAP (Fig.
CHX
tors (Fig. 2). has a profound effect on protein synthe- 5).
sis, preventing synthesisofthe majority ofthe protein bands The anti-LHCI we made to Porphyridium cruentum re-
labeled in the absence of inhibitor (Fig. 2, CON), whereas combinant LHCI recognized both the recombinant LHCI
the synthesis of many more labeled bands occurs in the antigen (Fig. 5A, lane 1) and the LHCI polypeptides from P.
presence of CAP. Furthermore, these protein bands differ cruentum thylakoids (Fig. 5B, lane 2). Six polypeptide
from those visualized in the CHX treatments (Fig. 2). bands were identified in P. cruentum, ranging in approxi-
mate molecularweights from 19to24kDa(Fig. 5B, lane 2),
Verification ofinhibitor effects LsiHzCesIcpoonlsyipsetpenttidewsitihn tthhiossesppercieevsiou(sTlayn detesaclr,ib1e9d95f)o.rTthhee
Cyt/antibodies reacted with a protein band synthesized antibody bound onto western blots of plastid proteins from
CHX
in the presence of on western blots at approximately Vciucheria litorea and Elysia chlorotica, with orwithout the
36 kDa (Fig. 3). Immunoprecipitations using anti-cyt / CHX and CAP treatments (Fig. 5C, lanes V. lit.. CON,
38 J. J. HANTEN AND S. K. PIERCE
CON CHX CAP Discussion
(kDa)
LHCI, a family ofplastid polypeptides essential for pho-
126- tosynthesis, is synthesized while Vaucheria litorea chloro-
plasts reside within the cells of the digestive diverticula of
90. Elysia chlorotica. In addition, our data indicate the LHCI
polypeptides are probably the products of genes located in
the host-cell nuclear genome because their synthesis is
inhibited by the cytosolic ribosome inhibitor, CHX, but not
by the presence ofthe plastid ribosome inhibitor, CAP. This
remarkable result would not be surprising in a plant oralgal
species since the LHCI polypeptide family's genes, Lhcal-
43.5 Lhca6, reside in the nuclear DNA of all plants and algae
examined to date (Jansson, 1994; Green and Durnford,
33.9_
B
(kDa)
126_
90
17.4
_
7.6 43.5
Figure2. AutoradiographofplastidproteinsseparatedbySDS-PAGE _
gelrununderthesameconditionsasthosedepictedinFigure2.Theplastid 33.9
proteins incorporating [35S]-methionine label differ following treatment
with CHX or CAP. The control (CON) represents chloroplast proteins
isolated from slugs without inhibitor treatment. Arrows identify the ap-
proximate positions of cyt/(large arrow) and the LHCI (small arrows)
17.4_
proteins.
CHX, CAP). As expected, the six polypeptide bands bound
by the anti-LHCI in V. litomi and E. chlorotica plastids
have a slightly greater size range 18 to 32 kDa than
those identified in P. cnicntiiin. These same antibody-la- _
7.6
beled bands from E. chlorotica plastid proteins incorporate
radioactive label in the presence of CAP. but incorporation
CHX
is blocked by the presence of (Fig. 6).
The amount ofradiolabel precipitated by anti-LHCI from
CHX
the slug plastid extracts following treatment is only
2% of the control level, indicating a reduction in LHCI
tsryenattheedsisslu(gFsigi.nc7)o.rpIonractoendtra[3s5tS,]t-hmeetLhHioCnIinperoatte9in2s%inofCcAoPn-- cproorFtrieegisnupsroenwde3ir.negaIluamtbmoeurlnaeddoiboalgsortadpehlsac(brBei)lb.eedTdhweiintshltuhgeasnmtweietbrohedoydesx.tpooAscneytdti-t/coyCt(AH/)X,biaannndddstihatets
trol rates, more than 40-fold higher than the level found in approximately 36 kDa, coincident with aradiolabeled protein. The arrow
CHX treated animals (Fig. 7). indicates the autorudiograph band corresponding to the position ofcyt/.
SYMBIOTIC PLASTID GENES IN SLUGS 39
CHX CAP
CONTROL^
(kDa)
16.8_
CBB Auto CBB Auto CBB Auto
Figure4. Immunoprecipitationofcyt/.Coomassiebrilliantblue(CBB)-stainedgelsofproteinsprecipitated
withanti-cyt/fromchloroplastextracts from slugssubjectedtonoinhibitor(Control),toCHX.ortoCAP.and
their corresponding autoradiographs (Auto). The arrow indicates the position ofcyt/. Large bands above and
below cyt/are the heavy and lightchainsofthe antibody, respectively. The radioactivitycorresponding to the
antibody bands in control and CHX is probably undissociated cyt/.
1996: Durnford et at., 1999: Jansson. 1999; Wollman eta/.. antibody, as well as all six ofthe native P. cnientum LHCI
1999). However, the synthesis of LHCI directed by an proteins (Tan etal., 1995; Grabowski etal., 2000) in control
animal's genome indicates that genes have been transferred immunoblots ofextracted thylakoids. The anti-LHCI immu-
into the slug DNA. noblots of E. chlorotica and V. litorea also identified six
Although surprising, the site of synthesis and the identi- protein bands with a greater size range than the LHCI
fication of LHCI seem to be without question as long as proteins identified in P. cnientum. Those bands are consis-
inhibitor and antibody specificity are not problems. Both tent with the sizes ofLHCI polypeptides from many species
CHX and CAP have been used in a wide array of studies, (Gantt. 1996; Jansson. 1999: Wollman et ai, 1999), and no
and their sites of action are well established. In fact, they other bands were labeled by the antibody. Seeing six LHCI
have been used, exactly as we have done here, to establish proteins is not surprising, because LHCI is typically found
that the site ofsynthesis ofthe "light harvesting chlorophyll in multiple homologs in algae, ranging from two in one
protein" (=LHCI) occurs on 80s cytoplasmic ribosomes in species ofXanthophyceae (Buchel and Wilhelm, 1993) toat
Phaeodactyliini tricomutum (Fawley and Grossman, 1986). least six paralogs in some rhodophytes (Tan et al., 1995),
There are several reasons to conclude that our antibody is and as many as eight in the chromophyte Heterosigma
specific. We raised the antibody against the red alga LHCI carterae (Durnford and Green, 1994). With few exceptions
not only because it was available, but also because the [such as in Euglena gracilis (Jansson, 1994)], each is en-
chromophytes, the taxonomic group of V. litorea, probably coded by a separate, nuclear gene belonging to the Lhc
arose through a secondary symbiosis from a red alga (Rieth, super-gene family (Jansson, 1999). Thus, location of the
1995; Green and Durnford, 1996; Palmer and Delwiche, gene aside, the presence of six LHCI proteins in the endo-
1996; Martin and Herrmann, 1998: Delwiche, 1999). Fur- symbiotic plastids in E. chlorotica is not surprising.
thermore. Porphyridium cnientum LHCI possesses both Itseemsclearthateach ofthe bands immunodecoratedby
sequence homologies and immunological relatedness to the anti-LHCI corresponds to a single LHCI polypeptide and
chromophytic light-harvesting proteins (Wolfe et ai, 1994; not a dimer. LHCI dimers can result from their association
Rieth, 1995; Tan etal.. 1997b). Thus, a polyclonal antibody with other LHC proteins and their respective photosystems
raised to a rhodophyte LHCI should have a good chance of /;; situ, and they do not always readily dissociate under the
specifically recognizing the LHCI polypeptides in V. lito- denaturing conditions of SDS-PAGE (Tan et al., 1995). If
rea. Our results indicate that the anti-LHCI binds the P. LHCI dimers were present here, they should have minimum
cnientum recombinant LHCI, the antieenic source for the molecular weights of about 36 kDa, corresponding to dou-
40 J. J. HANTEN AND S. K. PIERCE
A B C
CON CHX CAP
(kDa) 1 (kDa) 2 (kDa) V. lit
33.9
29.0
33.9
17.4 17.4
18.2
Figure5. Immunoblots testing the antibody raisedto Porphyridium inientum LHCI. (A) Anti-LHCI binds
the recombinant 22 kDaLlica RI product from P. cruentwn (lane i). Its appearance as a 28-30 kDaprotein in
SDS-PAGE and subsequent immunoblots results from the additionofa33 amino acid N-termina! fusion inthe
recombinant protein (Grabowski el at., 2000). (B) Anti-LHCI binds LHCI polypeptides extracted from P.
cruentumthylakoids (lane 2). (C) Vauclieria litorea (lane V. lit.)andEly\iachloroticaplastid proteinshavesix
bands binding the anti-LHCI identical in size toeach other. All six proteins are present in the slugs regardless
ofthe inhibitortreatment [lanesCON (control). CHX andCAP]. Molecularweights are indicatedtothe leftof
(A). (B). and (C).
ble the molecular weight of the smallest immunolabeled used here (Pierce et al.. 1996; Green et til., 2000). Further-
band. However, the largest ofthe six immunolabeled bands more, previous attempts to demonstrate FCPC synthesis
present in the gels is about 32 kDa, seemingly too small to with radioactive labels in the slugs have not yieldedpositive
be an LHCI dimer. results (Pierce et ai. 1996).
Other dimers might form with a number of photosystem The LHCII family of polypeptides is closely related to
I (PSD proteins due to the close association of LHCI with LHCI, performing similar functions in photosystem II to
the PSI subunits that compose the PSI-LHCI holocomplex those performed by LHCI in PSI. The LHC II genes are in
(Wollman et al.. 1999; Jansson. 1999). This also does not the same nuclear-encoded Lhc super-gene family (Jansson,
seem to be the case here. Anti-PSI. raised against the 1999) and share sequence homologies with those genes
cyanobacteria PSI holocomplex (again, courtesy of Profes- encoding LHCI (Durnford et al., 1999; Jansson. 1999;
sor Gantt), binds a single 10-kDa protein band on western Wollman et al., 1999). There is, however, aclear separation
blots ofE. chlorotica plastid proteins (data not shown). The in the phylogenies of LHCI and LHCII (Durnford et al..
combination of this PSI polypeptide with any of the three 1999), indicating some degree of dissimilarity between the
smaller bands (18-20 kDa) that react with the anti-LHCI two proteins. Nevertheless, the possibility seems to remain
could form a dimer with molecular weights comparable to that the proteins bound by our antibody could be from
each of the three larger polypeptides (28-32 kDa). How- LHCII.
ever, since anti-PSI and anti-LHCI do not co-label any Of the LHCII components, CP24. CP26, and CP29 con-
bands, an LHCI-PSI dimer is unlikely. tain the most sequence similarities to the LHCIs (Green and
An additional possibility might be that one of the bands Durnford. 1996) and have molecular weights. 25-30 kDa
could be another LHC-type protein possessing immunolog- (Wollman et al., 1999). that roughly correspond to these of
ical similarities to LHCI, such as the fucoxanthin chloro- the three largest polypeptides identified in our anti-LHCI
phyll ale binding proteins (FCPC) found in chromophytes immunoblots ofE. chlorotica and V. litorea plastid proteins
or light-harvesting complex II (LHCII) proteins. In fact, our (28-32 kDa), which appear to be slightly larger than most
previous work has demonstrated the presence of FCPC in LHC proteins inchromophytes (Green and Durnford, 1996).
plastids of both E. chlorotica and V. litorea. However, the An LHCII antibody derived from pea (generously donated
size of the FCPC protein identified there does not corre- by Dr. Kenneth Cline, University ofFlorida) was unreactive
spond to the weights ofthe proteins bound by the anti-LHCI in our iinmunoblotting protocol (data not shown). This
SYMBIOTIC PLASTID GENES IN SLUGS 41
CAP CHX
pare them. However, in a typical experiment, the values for
the amount of radioactive material incorporated into the
precipitate in the presence of CAP ranged from 5000 to
25,000cpm, whereasthose in the presenceofCHX ran from
150 to 400 cpm, which may give a clearer picture of the
level of material bound by the antibody.
The results of the pharmacological experiments, the im-
munoblots, and the immunoprecipitations. taken together,
provide substantial evidence that LHCI is the identity of
some of the plastid proteins that are synthesized in the
presence ofCAP. The inhibition ofLHCI synthesis by CHX
suggests that the algal Lhca genes have somehow been
transferred to the slug.
To be certain that a gene transfer has occurred, direct
evidence ofthe gene in the genomic DNA ofthe slug must
be found, and we are pursuing this confirmation. However,
in addition tothe results presented here,othercircumstantial
evidence for the transfer of the LHCI genes between alga
and slug is available in several characteristics of the asso-
ciation. First, although the turnover rate of LHCI in E.
chlomtica is unknown, the fact that it is synthesized indi-
cates that it is not an unusually robust protein LHCI
replacement is necessary for plastid function to proceed.
Second, Lhca genes have not been found in the plastid
genomes ofany organism (Durnford et /., 1999), including
other Vaucheria species (Linne von Berg and Kowallik,
1992). Of course, if LHCI were present in the plastid
genome, it would be synthesized with CHX present, as is
CAFPigaunrde365.[S]I-mmemtuhnioobnlinoet,(aInBd) iotfs cLoHrCrIespsoynndtihnegsiazeudtorinadtihoegrparpehse(nCcAeP)o.f the case with the cyt/controls; but it is not. Third, the V.
The arrows indicate radiolabeled bands coinciding to LHCI immunola- litorea plastid genome is 119.1 kb (Green et al., 2000),
beled bands shown in (IB). The bands in (CAP) are not labeled in the which is similar in size to those ofother algae, including V.
presence ofCHX (CHX).
125-
result seems to indicate that the polypeptides are not LHCII,
but since the similarity between the green plant and chro-
mophyte LHC proteins is relatively low (Green and Durn-
ford, 1996; Durnford et al., 1999), we probably cannot
completely eliminate the possibility that the anti-LHCI is
binding LHCII polypeptides. However, just like LHCI, all
of the LHCII genes are nuclear encoded in the plants and
algae where they have been found (Jansson, 1994, 1999;
Wollman et al.. 1999), and even if we have identified
LHCII, the conclusion is still the same: that an algal LHC
DNA
gene has been transferred to the of the slug.
The immunoprecipitations provide additional evidence
that the LHCI polypeptides are being synthesized on the
cytoplasmic ribosomes in the slug. The high amount of
radioactivity precipitated by the antibody in the presence of
CAP CHX
compared to that precipitated in the presence of
demonstrates that the proteins recognized by the anti-LHCI
are indeed synthesized in the slugs. Since the amount of
radioactivity incorporated varied from slug to slug and from
experiment to experiment, we had to normalize the immu-
noprecipitation data as percent of control in order to com-
42 J. J. HANTEN AND S. K. PIERCE
sessilisand V. hursata (Linne von Berg and Kowallik, 1988, of the chromophytic algae and their relatives. In many of
1992), hut small relative to those ofotherplants (Martin and thesecases, the identity ofthe initial host, symbiont, orboth
Herrmann. 1998). Even though the plastid genomesofchro- is unknown. In the case ofE. chlorotica and V. litorea, the
mophytic algae have a greater coding capacity, relative to origin of LHCI is known; if the gene has been transferred,
their size, than other algae because of fewer introns and the transfer occurred between two multicellular eukaryotes
inverted repeats (Rieth, 1995). they are too small to carry and represents a case of tertiary endosymbiosis.
sufficient genetic information to encode all of the enzymes Finally, the mechanism by which such a gene transfer
required for photosynthesis and plastid protein targeting. could occur may be found in the viruses that appear in each
DNA
Fourth, transfer of algal remnants or a nucleomorph- generation of the slugs at the end of their life cycle. The
type structure during plastid capture seems unlikely. To viruses have several features in common with Retroviridae
date, nucleomorphs have been found only in the Crypto- and seem to be endogenous (Pierce et al.. 1999). Retrovi-
phyta and Chlorarachniophyta (Delwiche. 1999; Zauner et ruses are capable of transferring genes between organisms;
til.. 2000) and have not been identified in any chromophyte if they are incorporated in the germ cells, they are trans-
DNA
(Maiereta!., 1991; Delwiche, 1999). Although ofthis ferred to the subsequent generations as Mendelian genes
type would probably be transcribed on nucleomorph SOS (Scharfman et al.. 1991). Thus, resolving the relationships
ribosomes (Douglas et al, 1991) and blocked by CHX. between the slugs, alga, plastids, and viruses may have
neither substantial electron microscopy (Kawaguti and Ya- profound implications for both cell and evolutionary biol-
masu, 1965; Graves et til., 1979; Mujer et al., 1996) nor ogy.
molecular testing (Green et al., 2000) has so far produced
evidence foreither nucleomorphs or algal nuclear remnants
DNA Acknowledgments
in E. chlorotica. Furthermore, if algal remnants were
present somewhere in the slugcells, the likelihood is remote Research support was provided by a National Science
oftheircontaining the correct genes and being present in all Foundation award (IBN-9604679) to SKP. We thank Elisa-
of the plastid-containing cells in all of the slugs in the beth Gantt and Beatrice Grabowski for their helpful sug-
populations year after year. Finally, others have suggested gestions.
that some of the proteins necessary to maintain photosyn-
thesis may be encoded in the mitochondria! genome and are
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