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In vitro synthesis of peroxisomal membrane polypeptides
Vol. 137, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 13, 1986
Pages
626-632
IN VITRO SYNTHESISOF PERDXISCZGG MEMBRANEPOLYPEPTIDES Andrea KZjster, Michael Heisig +, Peter C. Heinrich +, and Wilhelm W.Just Institute + Institute
of Biochemistry I, University of Heidelberg, D- 6900 Heidelberg,FRC of Biochemistry, University of Freiburg, D-7800 Freiburg,
Received April
FRC
22, 1986
Summary: Peroxisanal membranescontaining predominantly integral peroxisane membranepolypeptides were obtained fran a highly purified peroxisomal fraction. FollaJing sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and. used to raise antibodies in rabbits. Cell - free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisamal membranesto reductive methylationn4C$radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisame membranepolypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable preseguences for these peroxiscmal membranepolypeptides. 0
1986 Academic
Introduction:
Press,
Inc.
To date little
is known about the mechanismsof manmalian
peroxisane biogenesis. Three different
models are presently
discussed in
the literature. 1.
Peroxiscmes frcm liver
and even those of other tissues may be derived
from the endoplasmic reticulum by a budding off process classical
(I).
This
view is supported by morphological data demonstrating
continuities
between peroxisanes and the endoplasmic reticulum,
noted, however, that other morphological observations failed peroxisane-endoplasmic reticulum continuities 2. New peroxisomes are formed by fission
It must be
in confirming
(2,3).
of preexisting
ones (4). This
model is based mainly on biochemical data demonstrating that proteins located in the matrix of peroxiscmes are synthesized on free polysanes and are not sequestered in the endoplasmic reticulum but enter preexisting peroxisanes post-translationally 3.
The first
two &els
(5-10).
are canbined in a third model (5) confirming that
peroxisane content proteins were synthesized on membranefree polyscmes 0006-291X186 $1.50 Copyright All rights
0 1986 by Academtc Press, of reproduction m any form
Inc. reserved.
626
BIOCHEMICAL
Vol. 137. No. 2, 1986
and were post-translationally
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
imported via receptors and characteristic
signal sequences. PeroxiscaMl transmembrane polypeptides receptors for signal sequences, hover, cotranslationally The biosynthetic
polypeptides intensively
were hypothesized to be
inserted into the rough endoplasmic reticulum. pathways
and the intracellular
located in different investigated
subcellular
topqenesis
compartments were
follm
different
compartment depending on their
by recently
matrix or membranelocation.
of peroxisckM1 membranepolypeptides establishing
of extremely purified
peroxiscmes (12).
the Studies on the
(PMPs)'were facilitated
a procedure for the isolation
rat liver
It
mechanismsdepending on the
compartment for which they are destined and within
biosynthesis
of many
during the last years (for a review see 11).
turned out that polypeptides cellular
including
of large quantities
By this meansreasonable
amounts of pure peroxisanal membraneswere obtained and could be analyzed with respect to their with first
polypeptide
composition. The present paper deals
results on the production of monospecific polyclonal
antisera against three major PMPsfrom rat liver. these antisera of the in vitro
rabbit
Furthermore, the use of
is demonstrated to ccxnpare the apparent molecular weights synthesized PMPswith their
respective
mature prcducts.
Materials and Methods: Production of antisera: Peroxisamas were isolated as described previously (12) except that the animals received in their normal laboratory chw 0.5 % clofibrate for 14 days to stimulate peroxisane proliferation. Peroxisonal membraneswere isolated as in (13) and subjected to SDS-PA& Zones containing distinct PMPswere cut from the gel. The proteins were extracted with O.lM ammoniumbicarbonate, 0.1% SDS, pH 8.3 and concentrated by lyophilization. Excess of SDSwas removed with 80% acetone, 5% ammonia. After dissolution of the residue in 2ml of 0.9% NaCl equal amounts of complete Freti's adjuvants (Behring, Marburg) were added. 'Ihe obtained emulsion was injected S.C. into a rabbit on multiple sites on the back. Injections were reseated 4 and 8 weeks afterwards using incomplete Freund's adjuvant at the 8 week injection. The following amount of the three proteins were used for immunization: 1600 pg of PMP69, 740 pg of PMP36, 570 pg of PMP22). Ten days after the last booster blood was taken frtxn the ear vein of the animals. 1 Parts of this work were presented at the joint UNEXD-IUH Workshop on "Peroxisanes and Their Metabolites in Cellular Functions" held in Zeist, The Netherlands, August 22-24, 1985. 2 Abbreviations: PMP, peroxiscmal membranepolypeptide; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis. 627
Vol. 137, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Gel electrophoresis and fluorography: SLE-PAGE was performed as described previously (12). Proteins were visualized by silver staining essentially as described in (14). I$35&athionin-labeled proteins were visualized by fluorography according to (15) using glacial acetic acid instead of dimethylsulfoxide as a solvent for the scintillator. Autoradiography was carried out with Kodak X-O-r& AR X-ray sheets. In vitro translation: Heterologous in vitro translation of isolated total RNA or m-RNA was performed in the rabbit reticulocyte lysate system essentially as described previously (16). Total RNA was isolated by guanidinium hydrochloride &action (17) and poly-A+-RNA was obtained by affinity chranatography on oligo-dT-cellulose (Sigma, Munich) as described in (18). In vitro translation was terminated by the addition of SD!3 to a final concentration of 4%. After boiling the samples for 5 min they were diluted with 14OmM NaCl, 2OmM Tris-HCl pH 7.5, 5mM EDTA, FIN methionin and 1% Triton X-100 to a final SDS-concentration of 0.1%. After the additon of antisera, normally about 10~1, and 100 units Trasylol per ml (Bayer, Leverkusen) samples were left at 4OC over night. The antigen-antibody canplexes were recovered from these solutions by the addition of 1Omg protein-A Sepharose (Pharmacia, Freiburg) after a 1 h incubation at roan temperature. The recovered proteins were carefully washed and subjected to SE-PAGE and fluorography. This was carried out Reductive methylation of peroxiscnml polypeptides: as described in (19) but modified for membrane proteins by adding to the methylation buffer 1% SE. PMPs were isolated by the addition of antisera and protein-A Sepharose as described above and subjected to SDS-PAGE (Fig.la).
Results:
By canparison
and membrane proteins
of the SDS-electropherographs
three major PMPs with apparent molecular
69, 36, and 22 kDa were identified specifically sodium
located
carbonate
as integral
There are several
(Fig.lb).
apparent molecular
(20). The other polypeptides
the matrix
Portion.
be integral To follow
with 100 IIN present
in
weights of 82, 78, 49, 42.5, ware recently
shawn to
the isolation
of the
were found to be present also in
these polypeptides
were not considered
to
membrane canponents. specifically
polypeptides of antibodies
translated
(21). Fig.2a
the synthesis
of PMP 69, 36 and 22 these
were used to raise antibodies in the obtained
using 1125-labeled were
'Iberefore,
extraction
process during
organelles
weights of
other polypeptides
and 28 kDa. The 42.5 and the 28 kDa polypeptides fran PMP 69 by a proteolytic
matrix
PMPs which were
in the membrane withstanding
the membrane exhibiting
derive
of peroxiscmal
protein-A.
antisera Total
in a nuclease treated
were cellular
rabbit
shaws the SDS-PAGE of the total 628
in rabbits.
The concentrations
determined as well
by imnunoblotting as @y-A+
reticulocyte
lysate
translation
products
RNA both
system
Vol.
137,
BIOCHEMICAL
No. 2, 1986
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
68kDa-
YbkDa-
Fime 1: (a) Separation of polypsptides of total paroxisames (T), percHsaM matrix (M) and membranes (Me) by SDS-PAGEin a 7-158 gradient slab gel. Proteins derived fran 4 ug of peroxisanes of were loaded onto the gel. (b) Total clofibrate fed animals peroxisanes and peroxisaml membranes (1 q of each) were radiolabeled by reductive methylation and the radiolabeled polypeptides separated by SDS-PAGEas in (a).The polypeptides were visualized by fluorography. ST = molecular weight stmdars. Phosphorylase b (94 kDa), bovine serm albumin (68 klk), ovalbmin (43
demonstrating
kDa),
cartcanhydrase
the synthesis
150 kDa and higher.
isolated
polypeptides.
kh)
and
lysozyme
of polypeptides
with
The proteins of interest
imnmologically.
imunoprecipitated
(30
(14.5
kDa).
molecular
i.e.
weights
up to
PMP69, 36 and 22 were
By the sane way these proteins were
from peroxiscmes containing [14-amthylated
Both the in vitro
translated
and the mature[IBC$labsled PMps
were subjected to SDS-PAGE.The results of these experiments are shown in Fig.Zb.
They demonstrate that within a certain
3-5 amino acids, all
three in vitro
limit
translated
of detectability
i.e.
polypsptides were
synthesized with the same size as the mature organelle-incorporated polypeptides.
No
cleavable prepieces were detected under these conditions.
Discussion: The synthesis of several peroxismal recently
investigated
by in vitro
translation 629
matrix polypeptides was
in the rabbit
reticulccyte
Vol.
137,
No. 2, 1986
BfOCHEMlCAL
1
AND
2
BIOPHYSJCAL
I
a
2
RESEARCH
3
4
5
COMMUNICATIONS
6
b
94kDa94kDa68-
433D-
2014,5-
Fiqure 2: (a) In vitro translation of @y-A+ RNAisolated from the livers of clofibrate fed rats in a cell-free protein synthesizing systemderived fron rabbit reticulccytes. The total translational products in two different concentrations were separated by SDS-PAGE. (b) Mature peroxisml membrane pclypeptides were radiolabeled by reductive methylation. By the use of monospecific polyclonal antisera the polypeptides with apparent molecular weights of 69, 36 and 22 I&I were inrnunolqically isolated. By the sameway the respective in vitro translational products of these pclypeptides were obtained. The apparent molecular weights of both the mature (lane I,3 and 5) and the in vitro synthesized (lane 2‘4 and 61 polypaptides were ccmpared by SDS-PAGE in a 7-15%slab gel. The polypeptides in (a) and (b) were visualized by fluorcqraphy. molecular weight standards were as in legend to figure 1 except soy bean trypsin inhibitor (20 k&l. lysate or the wheat gem protein
synthesizing
that all the investigated
enzymes i.e.
oxidase, the bifunctional
protein
system
catalase,
(5-11).
It turn&
out
urate oxidase, acyl-CoA
- enoyl-COA hydratase/3-hydroxyacyl-COA
dehydrogenase - were synthesized with the samesize as the mature proteins.
Only 3-ketoacyl-COA thiolase makes an exception in that it
synthesized in a fom 3000 dalton larger than its nature subunit
is
(9,111.
Such an exception has also been reported to occur in glyoxysanes where r&ate
dehydrogenase is synthesized as a larger precursor whereas
isocitrate
lyase and malate synthase are formed without N-terminal
extensions (22-24). Much less information integral
is available
PMP was recently
on the synthesis of PMps. The 22 kDa
shown to be synthesized with the sameapparent 630
Vol.
137,
8lOCHEMlCAL
No. 2, 1986
molecular
weight
the present
as the mature product
paper confirm
that
two other
PMPS with
also
synthesized
with
Preliminary system
studies
indicated
results
of yeast
without
using
striking
with
'Ihe lack
similarity
polypeptides
weights
liver
as well
the proteins
derived
as of rat studied
show
translational
outer
liver
on integral
of the mitochondrial
PMPs and mitochondrial
membrane
(27-30).
so far were synthesized
as the corresponding
preseguences
to the polypeptides
became inserted
in
of 69 and 36 kDa were
of the mitochondrial
the same size
membrane. Whether integral
presented
(26).
cells
of cleavable
The experiments
of PMP 69 and PMP 22 predominantly
are known for proteins
all
COMMUNICATIONS
on PMP 22 and in addition
a homologous rat
crassa
RESEARCH
as the mature polypeptides.
freepolyriboscmes
presequences
polypeptides.
(25).
molecular
the synthesis
to these papers
BIOPHYSICAL
findings
apparent
and Neurospora
According
these
the same size
that
occuredonmembrane Similar
AND
outer
mature PMPS is a outer
membrane
by the same mechanism is currently
investigated. &nu&&ment~ 'Ihe authors discussion and many editorial Dautsche Forschungsgemeinschaft
thank Dr.H.Schirnassek for his stimulating suggestions.This work was supported by the (Ju 166/l-2)
References 1.
Novikoff,
A.B.,
and Novikoff,
P.M.
(1982)
Ann.N.Y.Acad.Sci.
386,
138-152. 2. 3. 4. 5.
Fahimi, H-D., ad Yokota, S. (1981) In International Cell Biology (Schweiger, H.G. ed.) ~~-640-650, Springer-Verlag, New York. Gorgas, K. (1984) AnaLEmbryol. 169, 261-270. Lazaraw, P.B. (1980) Ann.N.Y.Acad.Sci. 343, 293-303. Robbi, M., and Lazarow, P.B. (1978) Proc.Natl.Acad.Sci.USA 75, 4344-4348.
6.
Goldman, B.M., and Blobel,
G.
(1978)
Prcc.Natl.Acad.Sci.USA
75,
5066-5070. 7.
a. 9. 10. 11. 12.
Miura, S., Tori, M., Takiguchi, M., Tatibana, M., Furuta, S., Miyazawa,S., and &shin&o, T. (1984) J.Biol.Chem. 259, 6397-6402. Rachubinski, R.A., Fujiki, Y., Mxtensen, R.M., and Lazarcw, P.B. (1984) J-Cell Biol. 99, 2241-2246. Fujiki, Y., Rachubinski, R.A., Mxtensen, R.M., and Iazarcw, P.B. (1985) Biochem.J. 226, 697-704. Funlta, s., Hashtito, T., Miura, S., Mori, M., and Tatibana, M. (1982) Bicchem.Biophys.Res.Comrmn. 105, 639-646. Heinrich, P.C. (1982) Rev.Physiol.Biochem.Pharmacol. 93, 115-187. Hartl, F-U., Just, W-W., K&ater, A., and Schimassek, H. (1985) Arch. Biochem. Biophys. 237, 124-134. 631
Vol. 137, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
18.
Y., Fcwler, S., Shio, H., Hubbard, A.L., and Lazarow, P.B. J.Cell Biol. 93, 103-110. Ansorge, W. (1983) In Electrophoresis '82, (Stathakos, D. ed.) pp.235- 242, Walter de Gruyter & Co. Berlin, New York. Bonner, W.M., and Laskey, R.A. (1974) Eur.J.Bicchem. 46, 83-88. Northemann,W., Andus, T., Gross, V., and Heinrich, P.C. (1983) Eur.J.Biochem. 46, 83-88. Gregori, C., Besmmd, C., Kahn, A., and Dreyfus, J.-C. (1982) Biochem. Biophys. Res. Camnun. 104, 369-375. Aviv, H., and bailer, P. (1972) Proc. Natl. Acad. Sci. USA 69,
19.
Wystup, G-M., and Nierhaus,
13.
Fujiki,
(1982)
14. 15. 16. 17.
7 408-I 412.
K.H.
(1979)
Methods Enzymol.
59,
776-782. 21.
Hartl, F-U., and Just, W.W. (1985) Biol.Chem.Hoppe-Seyler 366, 797. Pelham, R.B., and Jackson, R.J. (1976) Eur.J.Biochem. 67,
22.
Reizman, H., Weir,
20.
247-256.
E., Leaver, C.J., Titus, D.E., and Becker, W.M. Plant Physiol. 65, 40-46. zirmmmnn, R., and Neupert, W. (1980) Eur.J.Biochem. 112, (1980)
23. 24. 25. 26. 27.
225-233. Walk, R.A. and Hock, B. 636-643
30.
Biochem. Biophys.
Res. Cumnm.
81,
Fujiki, Y., Rachubinski, R.A., and Lazarow, P.B. (1984) Proc. Natl. Acad. Sci. USA 81, 7127-7131. K&ter, A., Hartl, F-U., and Just, W.W. (1985) Biol.Chem.HoppeSeyler 366, 814. Freitag, H., Jams, M., and Neupert, W. (1982) Eur.J.Biochein. 126,
28. 29.
(1978)
197-202.
J.Biol.Chem. 258, 3427-3430. Gasser, J.M., and Schatz, G. (1983) R.A., and Shore, G.C. Noel, C., Nicolaou, V., Argan, C., Rachubinski, (1985) Biochim.Biophys.Acta 814, 35-42. Shore, G.C., power, F., Bendayan, M., and Carignan, P. (1981) J.Biol.Chem. 256, 8761-8766.
632
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 13, 1986
Pages
626-632
IN VITRO SYNTHESISOF PERDXISCZGG MEMBRANEPOLYPEPTIDES Andrea KZjster, Michael Heisig +, Peter C. Heinrich +, and Wilhelm W.Just Institute + Institute
of Biochemistry I, University of Heidelberg, D- 6900 Heidelberg,FRC of Biochemistry, University of Freiburg, D-7800 Freiburg,
Received April
FRC
22, 1986
Summary: Peroxisanal membranescontaining predominantly integral peroxisane membranepolypeptides were obtained fran a highly purified peroxisomal fraction. FollaJing sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and. used to raise antibodies in rabbits. Cell - free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisamal membranesto reductive methylationn4C$radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisame membranepolypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable preseguences for these peroxiscmal membranepolypeptides. 0
1986 Academic
Introduction:
Press,
Inc.
To date little
is known about the mechanismsof manmalian
peroxisane biogenesis. Three different
models are presently
discussed in
the literature. 1.
Peroxiscmes frcm liver
and even those of other tissues may be derived
from the endoplasmic reticulum by a budding off process classical
(I).
This
view is supported by morphological data demonstrating
continuities
between peroxisanes and the endoplasmic reticulum,
noted, however, that other morphological observations failed peroxisane-endoplasmic reticulum continuities 2. New peroxisomes are formed by fission
It must be
in confirming
(2,3).
of preexisting
ones (4). This
model is based mainly on biochemical data demonstrating that proteins located in the matrix of peroxiscmes are synthesized on free polysanes and are not sequestered in the endoplasmic reticulum but enter preexisting peroxisanes post-translationally 3.
The first
two &els
(5-10).
are canbined in a third model (5) confirming that
peroxisane content proteins were synthesized on membranefree polyscmes 0006-291X186 $1.50 Copyright All rights
0 1986 by Academtc Press, of reproduction m any form
Inc. reserved.
626
BIOCHEMICAL
Vol. 137. No. 2, 1986
and were post-translationally
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
imported via receptors and characteristic
signal sequences. PeroxiscaMl transmembrane polypeptides receptors for signal sequences, hover, cotranslationally The biosynthetic
polypeptides intensively
were hypothesized to be
inserted into the rough endoplasmic reticulum. pathways
and the intracellular
located in different investigated
subcellular
topqenesis
compartments were
follm
different
compartment depending on their
by recently
matrix or membranelocation.
of peroxisckM1 membranepolypeptides establishing
of extremely purified
peroxiscmes (12).
the Studies on the
(PMPs)'were facilitated
a procedure for the isolation
rat liver
It
mechanismsdepending on the
compartment for which they are destined and within
biosynthesis
of many
during the last years (for a review see 11).
turned out that polypeptides cellular
including
of large quantities
By this meansreasonable
amounts of pure peroxisanal membraneswere obtained and could be analyzed with respect to their with first
polypeptide
composition. The present paper deals
results on the production of monospecific polyclonal
antisera against three major PMPsfrom rat liver. these antisera of the in vitro
rabbit
Furthermore, the use of
is demonstrated to ccxnpare the apparent molecular weights synthesized PMPswith their
respective
mature prcducts.
Materials and Methods: Production of antisera: Peroxisamas were isolated as described previously (12) except that the animals received in their normal laboratory chw 0.5 % clofibrate for 14 days to stimulate peroxisane proliferation. Peroxisonal membraneswere isolated as in (13) and subjected to SDS-PA& Zones containing distinct PMPswere cut from the gel. The proteins were extracted with O.lM ammoniumbicarbonate, 0.1% SDS, pH 8.3 and concentrated by lyophilization. Excess of SDSwas removed with 80% acetone, 5% ammonia. After dissolution of the residue in 2ml of 0.9% NaCl equal amounts of complete Freti's adjuvants (Behring, Marburg) were added. 'Ihe obtained emulsion was injected S.C. into a rabbit on multiple sites on the back. Injections were reseated 4 and 8 weeks afterwards using incomplete Freund's adjuvant at the 8 week injection. The following amount of the three proteins were used for immunization: 1600 pg of PMP69, 740 pg of PMP36, 570 pg of PMP22). Ten days after the last booster blood was taken frtxn the ear vein of the animals. 1 Parts of this work were presented at the joint UNEXD-IUH Workshop on "Peroxisanes and Their Metabolites in Cellular Functions" held in Zeist, The Netherlands, August 22-24, 1985. 2 Abbreviations: PMP, peroxiscmal membranepolypeptide; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis. 627
Vol. 137, No. 2, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Gel electrophoresis and fluorography: SLE-PAGE was performed as described previously (12). Proteins were visualized by silver staining essentially as described in (14). I$35&athionin-labeled proteins were visualized by fluorography according to (15) using glacial acetic acid instead of dimethylsulfoxide as a solvent for the scintillator. Autoradiography was carried out with Kodak X-O-r& AR X-ray sheets. In vitro translation: Heterologous in vitro translation of isolated total RNA or m-RNA was performed in the rabbit reticulocyte lysate system essentially as described previously (16). Total RNA was isolated by guanidinium hydrochloride &action (17) and poly-A+-RNA was obtained by affinity chranatography on oligo-dT-cellulose (Sigma, Munich) as described in (18). In vitro translation was terminated by the addition of SD!3 to a final concentration of 4%. After boiling the samples for 5 min they were diluted with 14OmM NaCl, 2OmM Tris-HCl pH 7.5, 5mM EDTA, FIN methionin and 1% Triton X-100 to a final SDS-concentration of 0.1%. After the additon of antisera, normally about 10~1, and 100 units Trasylol per ml (Bayer, Leverkusen) samples were left at 4OC over night. The antigen-antibody canplexes were recovered from these solutions by the addition of 1Omg protein-A Sepharose (Pharmacia, Freiburg) after a 1 h incubation at roan temperature. The recovered proteins were carefully washed and subjected to SE-PAGE and fluorography. This was carried out Reductive methylation of peroxiscnml polypeptides: as described in (19) but modified for membrane proteins by adding to the methylation buffer 1% SE. PMPs were isolated by the addition of antisera and protein-A Sepharose as described above and subjected to SDS-PAGE (Fig.la).
Results:
By canparison
and membrane proteins
of the SDS-electropherographs
three major PMPs with apparent molecular
69, 36, and 22 kDa were identified specifically sodium
located
carbonate
as integral
There are several
(Fig.lb).
apparent molecular
(20). The other polypeptides
the matrix
Portion.
be integral To follow
with 100 IIN present
in
weights of 82, 78, 49, 42.5, ware recently
shawn to
the isolation
of the
were found to be present also in
these polypeptides
were not considered
to
membrane canponents. specifically
polypeptides of antibodies
translated
(21). Fig.2a
the synthesis
of PMP 69, 36 and 22 these
were used to raise antibodies in the obtained
using 1125-labeled were
'Iberefore,
extraction
process during
organelles
weights of
other polypeptides
and 28 kDa. The 42.5 and the 28 kDa polypeptides fran PMP 69 by a proteolytic
matrix
PMPs which were
in the membrane withstanding
the membrane exhibiting
derive
of peroxiscmal
protein-A.
antisera Total
in a nuclease treated
were cellular
rabbit
shaws the SDS-PAGE of the total 628
in rabbits.
The concentrations
determined as well
by imnunoblotting as @y-A+
reticulocyte
lysate
translation
products
RNA both
system
Vol.
137,
BIOCHEMICAL
No. 2, 1986
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
68kDa-
YbkDa-
Fime 1: (a) Separation of polypsptides of total paroxisames (T), percHsaM matrix (M) and membranes (Me) by SDS-PAGEin a 7-158 gradient slab gel. Proteins derived fran 4 ug of peroxisanes of were loaded onto the gel. (b) Total clofibrate fed animals peroxisanes and peroxisaml membranes (1 q of each) were radiolabeled by reductive methylation and the radiolabeled polypeptides separated by SDS-PAGEas in (a).The polypeptides were visualized by fluorography. ST = molecular weight stmdars. Phosphorylase b (94 kDa), bovine serm albumin (68 klk), ovalbmin (43
demonstrating
kDa),
cartcanhydrase
the synthesis
150 kDa and higher.
isolated
polypeptides.
kh)
and
lysozyme
of polypeptides
with
The proteins of interest
imnmologically.
imunoprecipitated
(30
(14.5
kDa).
molecular
i.e.
weights
up to
PMP69, 36 and 22 were
By the sane way these proteins were
from peroxiscmes containing [14-amthylated
Both the in vitro
translated
and the mature[IBC$labsled PMps
were subjected to SDS-PAGE.The results of these experiments are shown in Fig.Zb.
They demonstrate that within a certain
3-5 amino acids, all
three in vitro
limit
translated
of detectability
i.e.
polypsptides were
synthesized with the same size as the mature organelle-incorporated polypeptides.
No
cleavable prepieces were detected under these conditions.
Discussion: The synthesis of several peroxismal recently
investigated
by in vitro
translation 629
matrix polypeptides was
in the rabbit
reticulccyte
Vol.
137,
No. 2, 1986
BfOCHEMlCAL
1
AND
2
BIOPHYSJCAL
I
a
2
RESEARCH
3
4
5
COMMUNICATIONS
6
b
94kDa94kDa68-
433D-
2014,5-
Fiqure 2: (a) In vitro translation of @y-A+ RNAisolated from the livers of clofibrate fed rats in a cell-free protein synthesizing systemderived fron rabbit reticulccytes. The total translational products in two different concentrations were separated by SDS-PAGE. (b) Mature peroxisml membrane pclypeptides were radiolabeled by reductive methylation. By the use of monospecific polyclonal antisera the polypeptides with apparent molecular weights of 69, 36 and 22 I&I were inrnunolqically isolated. By the sameway the respective in vitro translational products of these pclypeptides were obtained. The apparent molecular weights of both the mature (lane I,3 and 5) and the in vitro synthesized (lane 2‘4 and 61 polypaptides were ccmpared by SDS-PAGE in a 7-15%slab gel. The polypeptides in (a) and (b) were visualized by fluorcqraphy. molecular weight standards were as in legend to figure 1 except soy bean trypsin inhibitor (20 k&l. lysate or the wheat gem protein
synthesizing
that all the investigated
enzymes i.e.
oxidase, the bifunctional
protein
system
catalase,
(5-11).
It turn&
out
urate oxidase, acyl-CoA
- enoyl-COA hydratase/3-hydroxyacyl-COA
dehydrogenase - were synthesized with the samesize as the mature proteins.
Only 3-ketoacyl-COA thiolase makes an exception in that it
synthesized in a fom 3000 dalton larger than its nature subunit
is
(9,111.
Such an exception has also been reported to occur in glyoxysanes where r&ate
dehydrogenase is synthesized as a larger precursor whereas
isocitrate
lyase and malate synthase are formed without N-terminal
extensions (22-24). Much less information integral
is available
PMP was recently
on the synthesis of PMps. The 22 kDa
shown to be synthesized with the sameapparent 630
Vol.
137,
8lOCHEMlCAL
No. 2, 1986
molecular
weight
the present
as the mature product
paper confirm
that
two other
PMPS with
also
synthesized
with
Preliminary system
studies
indicated
results
of yeast
without
using
striking
with
'Ihe lack
similarity
polypeptides
weights
liver
as well
the proteins
derived
as of rat studied
show
translational
outer
liver
on integral
of the mitochondrial
PMPs and mitochondrial
membrane
(27-30).
so far were synthesized
as the corresponding
preseguences
to the polypeptides
became inserted
in
of 69 and 36 kDa were
of the mitochondrial
the same size
membrane. Whether integral
presented
(26).
cells
of cleavable
The experiments
of PMP 69 and PMP 22 predominantly
are known for proteins
all
COMMUNICATIONS
on PMP 22 and in addition
a homologous rat
crassa
RESEARCH
as the mature polypeptides.
freepolyriboscmes
presequences
polypeptides.
(25).
molecular
the synthesis
to these papers
BIOPHYSICAL
findings
apparent
and Neurospora
According
these
the same size
that
occuredonmembrane Similar
AND
outer
mature PMPS is a outer
membrane
by the same mechanism is currently
investigated. &nu&&ment~ 'Ihe authors discussion and many editorial Dautsche Forschungsgemeinschaft
thank Dr.H.Schirnassek for his stimulating suggestions.This work was supported by the (Ju 166/l-2)
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