Protein transport into canine pancreatic microsomesA quantitative approach Silvia Guth*, Christian Vo¨lzing*, Anika Mu¨ller, Martin Jung and Richard Zimmermann Medizinische Biochemie und
Trang 1Protein transport into canine pancreatic microsomes
A quantitative approach
Silvia Guth*, Christian Vo¨lzing*, Anika Mu¨ller, Martin Jung and Richard Zimmermann
Medizinische Biochemie und Molekularbiologie, Universita¨t des Saarlandes, Homburg, Germany
Transport of presecretory proteins into the mammalian
rough endoplasmic reticulum involves a protein translocase
that comprises the integral membrane proteins Sec61ap,
Sec61bp, and Sec61cp as core components Electron
microscopic analysis of protein translocase in rough
micro-somal membranes suggested that between three and four
heterotrimeric Sec61p complexes form the central unit of
protein translocase Here we analyzed the stoichiometry of
heterotrimeric Sec61p complexes present in cotranslationally
active protein translocases of canine pancreatic microsomes and various other lumenal and membrane components believed to be subunits of protein translocase and to be involved in covalent modifications Based on these numbers, the capacity for cotranslational transport was estimated for the endoplasmic reticulum of the human pancreas Keywords: endoplasmic reticulum; mammalian microsomes; protein secretion; protein transport; pancreas
Transport of presecretory proteins into mammalian rough
microsomes involves cleavable signal peptides at the
N-terminus of the precursor proteins and a protein
translo-case in the microsomal membrane [1] Typically, transport
occurs as a sequence of three consecutive steps, namely (a)
specific membrane association of the precursor protein (also
termed targeting), (b) membrane insertion, and (c)
comple-tion of translocacomple-tion Specific membrane associacomple-tion of
precursors in cotranslational transport involves two
ribonu-cleoparticles – the ribosome [2] and the signal recognition
particle (SRP) [3] – as well as their receptors on the
endoplasmic reticulum (ER) surface (the SRP and ribosome
receptors) [4,5] Protein translocase (a) mediates both
membrane insertion and completion of translocation, (b)
comprises Sec61ap, Sec61bp, and Sec61cp [5] as core
components, and (c) operates co- or post-translationally In
addition, heterotrimeric Sec61p complexes serve as specific
ribosome-binding sites in cotranslational transport [6], and
as signal peptide receptors in general [7] The concentration
of heterotrimeric Sec61p complexes and specifically bound
ribosomes in defined suspensions of mammalian microsomes
(absorbance¼ 50 at 280 nm in 2% SDS, corresponding to 1
equivalent per lL) has been determined as 1.67–2.12 lM[8,9]
and 0.27–0.39 lM[6,8], respectively These data were taken
as a first suggestion that oligomers of heterotrimeric Sec61p
complexes may be associated with ribosomes that are
simultaneously engaged in protein synthesis and transloca-tion Subsequently, cryo- and freeze fracture electron micro-scopic analysis of the Sec61p complexes, as present in intact membranes, derived from canine pancreatic or yeast endo-plasmic reticulum, suggested that between three and four Sec61p complexes form the central unit of the protein translocase [8,10–12] In addition to the heterotrimeric Sec61p complexes, Hsp70 protein family members of the
ER lumen (BiP/Grp78 and Grp170) are part of the protein translocase and facilitate insertion of presecretory proteins into the Sec61p complex, as well as completion of translo-cation [13,14] These Hsp70 protein family members of the mammalian ER may be recruited to the Sec61p complex by the membrane-integrated Hsp40 protein family members, Sec63p [9,15] and/or Mtj1p [16] Sec62p [9,15], TRAMp [17], and the TRAP complex [18] appear to be additional subunits
of protein translocase Many precursor proteins that enter the ER are processed by the signal peptidase complex [19] and the oligosaccharyl transferase complex [20] Therefore, it
is not surprising that these complexes are in close proximity
to protein translocase [21,22] When misfolding occurs, the polypeptides are exported to the cytosol and degraded by the proteasome Protein export from the ER lumen to the cytosol
is also mediated by Sec61p complexes [23,24]
Here we addressed the stoichiometry of the mammalian Sec61p complexes that are present in cotranslationally active protein translocases, by quantitative analysis of protein transport into pancreatic microsomes in single turnover translocation experiments, and of various other components, believed to be subunits of protein translocase and involved in covalent modifications, by semiquantitative immunoblot analysis
Experimental procedures
Materials The luciferase assay reagent and anti-luciferase immuno-globulin were obtained from Promega The Translation kit
Correspondence to R Zimmermann, Medizinische Biochemie und
Molekularbiologie, Universita¨t des Saarlandes, D-66421 Homburg,
Germany Fax: +49 6841 1626288; Tel.: +49 6841 1626510;
E-mail: bcrzim@med-rz.uni-saarland.de
Abbreviations: BiP, immunoglobulin heavy chain binding protein;
ECL, enhanced chemiluminescence; ER, endoplasmic reticulum;
ppl, preprolactin; PVDF, poly(vinylidene difluoride); SRP, signal
recognition particle; SR, SRP receptor.
*These authors contributed equally to this work.
(Received 2 February 2004, revised 11 May 2004,
accepted 10 June 2004)
Trang 2type II and firefly luciferase were from Roche Diagnostics.
The peroxidase conjugate of goat antirabbit IgG was from
Sigma Chemical Company [35S]Methionine, X-ray films
and enhanced chemiluminescence (ECL) reagents were
from Amersham Biosciences; poly(vinylidene difluoride)
(PVDF) membranes were from Millipore
In vitro translation/translocation
Protein synthesis was carried out in rabbit reticulocyte
lysates in the presence of [35S]methionine, following the
supplier’s recommendations (Translation kit type II; Roche
Diagnostics) Subsequently, the samples were subjected to
SDS/PAGE The dried gels were analyzed in a
phosphor-imager (Molecular Dynamics, Sunnyvale, CA, USA) using
IMAGEQUANTsoftware (version 5.1, Molecular Dynamics)
Alternatively, the proteins were transferred to PVDF
membranes and incubated with specific antibodies The
antibodies were visualized by ECL and subsequent exposure
to X-ray film X-ray films were analyzed by densitometry
(Molecular Dynamics) using IMAGEQUANT software
(ver-sion 3.0; Molecular Dynamics) Images from
phosphori-mager and densitometry analyses were transferred into
PHOTOSHOPsoftware [version 3.0.5, Adobe Systems, Inc.,
San Jose, CA, USA] for production of figures Luciferase
activity was determined as described previously [25]
Quantification of proteins synthesizedin vitro
Firefly luciferase was used as an endogenous reference for
the quantification of radiolabeled proteins This is possible
because luciferase, newly synthesized in rabbit reticulocyte
lysate, is folded to its native state with a very high efficiency
and reproducibility In a first set of experiments, serial
dilutions of purified luciferase in reticulocyte lysate were
subjected to luciferase activity measurements as well as to
immunoblot analyses The blot was incubated with
anti-luciferase immunoglobulin and a peroxidase conjugate of
secondary antibodies The antibodies were visualized by
ECL and subsequent exposure to X-ray film The films were
analyzed by densitometry Both data sets gave rise to
standard curves that served as a reference for unknown
quantities of luciferase in subsequent experiments (data not
shown) In a second set of experiments, luciferase was
synthesized in reticulocyte lysate in the presence of
[35S]methionine for 60 min Subsequently, luciferase activity
and radioactivity, present in the luciferase band after SDS/
PAGE, were determined for different aliquots of the same
translation reaction by luminometry and phosphorimager
analysis, respectively In addition, different aliquots of the
luciferase translation reactions were subjected to SDS/
PAGE and subsequent blotting to a PVDF membrane,
together with serial dilutions of purified luciferase The blot
was analyzed as described above Based on the two
above-mentioned standard curves, the quantity of newly
synthesized luciferase was determined In both analyses,
calculations were based on data points that lay in the linear
range of the standard curves The results from both analyses
led to the conclusion that the concentration of de
novo-synthesized luciferase in the reticulocyte lysate is 2 nM
after in vitro translation for 60 min, i.e for the batches used
of reticulocyte lysate and [35S]methionine Subsequently, the
quantity of a given protein, after synthesis in the same batch
of an in vitro system, was determined, with reasonable accuracy, by phosphorimager analysis of the respective gel band and by its comparison with simultaneously synthes-ized firefly luciferase (assayed by both luminometry and phosphorimaging) In these experiments, the luciferase activity and the radioactivity analysis allowed the calcula-tion that the concentracalcula-tion of a nascent preprolactin polypeptide chain (ppl-86mer, four methionines) and full-length preprolactin (ppl, eight methionines) in the in vitro system were 250 nM and 100 nM, respectively, after translation for 20 and 45 min, respectively The different methionine contents of luciferase (13 methionines) and the other proteins was taken into account
Quantification of microsomal proteins Dog pancreas microsomes were prepared and treated with nuclease and EDTA, as described previously [26] The absorbance at 280 nm, in 2% SDS, of the final microsomal suspension was 50, corresponding to 1 equivalent per lL, or a protein concentration of 15 mgÆmL)1 The Sec61p com-plex, SRP receptor, TRAMp, TRAP comcom-plex, signal peptidase complex and oligosaccharyl transferase complex were purified according to previously published procedures [5,17,18,20] and used for quantification according to our previously published procedure [9] Briefly, the quantity of protein, present in the respective band of a gel of a certain protein preparation, was determined by comparison with protein standards that were run on the same gel and stained simultaneously with Coomassie Brilliant Blue Subsequently,
an aliquot of the same sample of purified protein was run on the same gel together with increasing amounts of micro-somes, and the known quantity of purified protein served as a standard for the Western blot signals, as determined by luminescence and densitometry of the X-ray films In both analyses, calculations were based on data points that lay in the linear range of the densitometry signals We note that the calculations are based on the assumption that staining with Coomassie Brilliant Blue is uniform for all proteins and that this may not be absolutely true for every single protein Therefore, the values that are given in Table 1 should be interpreted with this caveat in mind
Results
Capacity of canine pancreatic microsomes for SRP-dependent protein transport
The ability to quantify newly synthesized proteins based on radioactivity analyses and comparison with simultaneously synthesized firefly luciferase (as described in the Experi-mental procedures) allowed us to analyze the efficiency of protein transport into canine pancreatic microsomes at different ratios of the precursor and Sec61ap Single SRP– ribosome–nascent preprolactin chain (ppl-86mer) com-plexes were produced in the presence of increasing concen-trations of pancreatic microsomes This experimental strategy is defined here as a single turnover experiment and was first described by Connolly & Gilmore [27] Subsequently, the translation reactions were divided into four aliquots One aliquot was untreated (Fig 1A) and used
Trang 3for determining the total quantity of ppl-86mer in the
translation reaction Microsomes were reisolated from the
other three aliquots and untreated (Fig 1B), or subjected to
puromycin-induced translocation of the nascent
presecre-tory protein (termed chase) (Fig 1C), or subjected to
chemical cross-linking (Fig 1D) Luciferase was synthesized
in parallel and quantified on the basis of its enzymatic
activity After SDS/PAGE and phosphorimager analysis,
the precursor, mature protein and cross-linked precursor
were quantified in comparison to luciferase analyzed
simultaneously (as described in the Experimental
proce-dures) The quantities (a) of total ppl-86mer (Fig 1A,E),
synthesized in the translation reaction (b) of
microsome-bound ppl-86mer (Fig 1B,F), (c) of chased, i.e specifically
bound ppl-86mer (Fig 1C,G), and (d) of
Sec61ap-associ-ated, i.e cross-linked, ppl-86mer (Fig 1D,H), were
com-pared with the quantities of Sec61p complexes [9] present in
the various translation reactions According to the efficien-cies of ppl-86mer synthesis, the ratios between precursor and Sec61ap varied between 12.5 and 0.4 (Fig 1E, e.g the
Table 1 Concentration of components in pancreatic rough microsomes (RM) In the case of complexes, the proteins, shown in parentheses, were quantified Note that the concentrations refer to a suspension of
RM with an absorbance at 280 nm of 50, as measured in 2% SDS, corresponding to 1 equivalent per lL, or a protein concentration of
15 mgÆmL)1or 0.33 m M (average molecular mass 50 000 kDa) BiP, immunoglobulin heavy chain binding protein; OST, oligosaccharyl transferase; SPase, signal peptidase; SRP, signal recognition particle Component Concentration Reference High-affinity ribosome-binding sitesa 0.27–0.39 l M [6,8] Cotranslationally operating
translocases b
0.40–0.62 l M
SRP receptor (SRap) 0.24 l M
SRP receptor (SRbp) 0.47 l M
Sec61p complex (Sec61ap) 2.12 l M [9]
TRAP complex 1.30 l M [31] Sec62p 1.96 l M [9] Sec63p 1.98 l M [9] Mtj1p 0.36 l M [16]
Grp170 0.60 l M [30] SPase complex (SPC23-su) 0.52 l M
OST complex (Ost48p) 1.60 l M
a
High salt resistant ribosome-binding sites at a concentration of Sec61 ap of 1.67 l M bProductive binding sites for SRP–ribosome– nascent chain complexes; as deduced from Figs 1 and 3, respect-ively; average values.
Fig 1 Quantification of specific binding of nascent presecretory pro-teins to microsomes and Sec61ap in single turnover experiments Nas-cent preprolactin (ppl-86mer) was synthesized in reticulocyte lysates in the presence of [ 35 S]methionine and dog pancreas microsomes at the indicated concentrations [rough microsomes (RM), %, v/v] After incubation for 20 min at 30 C, the translation reactions were divided into four aliquots (A–D) One aliquot was untreated (A), and aliquots 2–4 were subjected to centrifugation (20 min, 15 000 g, 2 C) (B–D) The pellet from the second aliquot was untreated thereafter (B) The pellet from the third aliquot was resuspended in buffer and incubated for 15 min at 30 C in the presence of puromycin (1.25 m M ) (C) The pellet from the fourth aliquot was resuspended in buffer and incubated with 335 l M succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carb-oxylate (SMCC) for 20 min at 0 C, as described previously [13] (D) Firefly luciferase was synthesized in parallel and luciferase activity was assayed Different aliquots of all samples (including the luciferase translation reaction) were subjected to SDS/PAGE (19.4% acrylamide + urea) and phosphorimager analysis The quantification of ppl-86mer was carried out as described in the Experimental procedures (E–H) The data for 1 lL (A) and 5 lL (B–D) aliquots are shown The dotted line in (F) represents the sum of ppl-86mer and ppl-86mer cross-linked to Sec61ap of (C), i.e allows an estimation of the protein recovery after cross-linking Note that the different electrophoretic mobility of ppl-86mer in lane 4 of (B) is caused by a gel artifact ppl 86 xSec61ap, ppl-86mer cross-linked to Sec61ap; ppl 86 , ppl-86mer;
pl , pl-56mer.
Trang 4first data point: 250 nmol of ppl-86mer divided by 20 nmol
of Sec61ap corresponds to a ratio of 12.5 : 1.0) The binding
of ppl-86mer to microsomes (Fig 1F) and the specific
binding of ppl-86mer to microsomes (Fig 1G), measured as
chase to pl-56mer, increased with increasing concentration
of microsomes As expected, at the lowest concentration of
microsomes, the total binding exceeded by far the specific
binding [28] (Fig 1F vs 1G) However, the specific binding
was significant at higher concentrations of microsomes
(Fig 1F vs 1G) There was a good correlation between
specific binding and cross-linking to Sec61ap (Fig 1G vs
1H) and cross-linking of specifically bound ppl-86mer was
quite efficient (up to 80%; Fig 1G vs 1H) For the two
intermediate concentrations of microsomes (4 and 8% RM,
respectively, i.e at 25- and 12.5-fold dilutions) the results,
shown in Fig 1, indicate concentrations of specific binding
sites for ribosome–nascent chain complexes of ppl-86mer in
the microsomal suspension between 0.34 (Fig 1H, e.g the
second data point/80 nmol Sec61ap: 15 nmol of
ppl-86mer· 25 ¼ 375 nmol) and 0.46 lM (Fig 1G, e.g the
third data point/160 nmol Sec61ap: 38 nmol
ppl-86mer· 12.5 ¼ 475 nmol) (average: 0.4 lM; Table 1) (note
that the concentration of Sec61ap in the microsomal
suspension is 2 lM; Table 1) As cross-linking typically
does not occur at efficiencies of 100%, the latter number
seems to be more reliable Thus, at these intermediate
concentrations of microsomes, the average ratio between
specifically bound ppl-86mer and Sec61ap was 1.0 : 4.3
(Fig 1G; 0.46 /2 lMcorresponds to a ratio of 1.0 : 4.3), i.e
about one in four Sec61ap molecules was able to bind the nascent precursor protein Similar experiments were carried out employing rough microsomes that had been pretreated with puromycin plus high salt and yielded similar results (data not shown) This must be caused by read-out synthesis
on ribosomes that were attached to microsomes in the mammalian translation system
In order to confirm the notion that the conditions of the single turnover experiment allow saturation of specific binding, a two-stage experiment was carried out The first stage of this experiment was similar to the single turnover experiment, but was carried out in the absence of [35S]methionine and at an intermediate concentration of microsomes (5% RM, v/v) Subsequently, microsomes were reisolated and subjected to a second translation reaction in the presence of [35S]methionine, i.e with or without prior EDTA treatment In parallel, microsomes were subjected to
a first mock translation reaction in the absence of transcript and [35S]methionine and, after reisolation and treatment with or without EDTA, to a second translation reaction in the presence of [35S]methionine The precursor preprolactin was synthesized in the second stage of the experiment, and the various microsomes were present in these translation reactions at two different final concentrations (5 or 10%
RM, v/v; Fig 2A) A control experiment for this second
Fig 2 Specific binding of nascent presecretory proteins to microsomes
in a single turnover experiment prevents cotranslational transport of
other presecretory proteins in a subsequent transport reaction Nascent
preprolactin (ppl-86mer) was synthesized in reticulocyte lysate in the
presence of dog pancreas microsomes [5% rough microsomes (RM),
v/v) (+mRNA)] A mock translation minus transcript was carried out
in parallel (–mRNA) After incubation for 20 min at 30 C, the
translation reactions were centrifuged (20 min, 15 000 g, 2 C) The
pellets were resuspended in buffer, divided into two aliquots, and
incubated in the absence (–) or presence (+) of EDTA (5 m M ) for
30 min at 30 C MgCl 2 (7.5 m M ) was added to all samples which were
then adjusted to the same final concentration of EDTA Subsequently,
preprolactin was synthesized in reticulocyte lysates in the presence of
[ 35 S]methionine and in the simultaneous presence of the different
microsomes at the indicated concentrations (5 or 10% RM, v/v;
stip-pled vs solid bars in C) (A,C) In parallel, preprolactin was synthesized
in rabbit reticulocyte lysates in the presence of [ 35 S]methionine and in
the simultaneous presence of dog pancreas microsomes at the indicated
concentrations (RM, %, v/v) (B) After incubation for 45 min at
30 C, the translation reactions were supplemented with puromycin
and incubated further for 15 min at 30 C All samples were subjected
to SDS/PAGE (19.4% acrylamide+urea) and phosphorimager
ana-lysis (A–C) Note that radiolabeled ppl-86mer was synthesized in the
second translation reaction as a result of the presence of mRNA which
was carried over from the first translation reaction (A) The other
presecretory proteins were analyzed in a similar manner (D,E) or under
post-translational transport conditions (F), as described previously
[29] ppl, preprolactin; ppl 86 , ppl-86mer; pl, prolactin; pl 56 ,
pl-56mer; ppaf, prepro-a-factor; ppcecDHFR,
preprocecropin-dihydrofolate reductase hybrid protein.
Trang 5translation confirmed that the concentrations of
micro-somes that were used in the second stage of the two-stage
experiment allowed the detection of quantitative differences
in transport efficiencies (Fig 2B) After SDS/PAGE,
phos-phorimager analysis of precursor and mature protein was
carried out (Fig 2A,C) Transport of preprolactin was
almost completely blocked when microsomes were analyzed
which had previously been subjected to a single turnover
experiment with ppl-86mer (Fig 2A,C, lanes/bars 3 and 4)
This effect is most obvious when the two concentrations of
microsomes are compared (Fig 2A,C, lanes/bars 3 vs 4)
However, transport of preprolactin was only minimally
affected when microsomes were analyzed which had
previ-ously been subjected to a single turnover experiment and,
subsequently, to a chase of ppl-86mer with EDTA
(Fig 2A,C, lanes/bars 1 and 2) Furthermore, microsomes
were minimally affected by the first mock translation
(Fig 2A,C, lanes/bars 5–8) Thus, the two-stage experiment
demonstrated that the single turnover experiments had led
to saturation of microsomes with respect to their transport
capacity This was confirmed by employing, in the two-stage
experiment, a second precursor that is transported in an
SRP-dependent manner and cotranslationally, yeast
pre-pro-a-factor (Fig 2D, bars 3 and 4 vs 1 and 2), and a
precursor that is transported predominantly in an
SRP-dependent manner and cotranslationally when it is
synthes-ized in the presence of microsomes, a preprocecropin–
dihydrofolate reductase hybrid (Fig 2E, bars 3 and 4 vs 1
and 2) [29] We note that yeast prepro-a-factor is
transpor-ted into mammalian microsomes only cotranslationally and
that the preprocecropin–dihydrofolate reductase hybrid
is transported into mammalian microsomes both
co-and post-translationally under cotranslational conditions
(Fig 2E) and, obviously, only post-translationally under
post-translational conditions (Fig 2F) [29]
SRP-independ-ent and post-translational transport of the preprocecropin–
dihydrofolate reductase hybrid was not affected by
satura-tion of microsomes with respect to their cotranslasatura-tional
transport capacity (Fig 2F) We note that the observation
that the preprocecropin–dihydrofolate reductase hybrid
under cotranslational conditions was affected less than
preprolactin and the prepro-a-factor (Fig 2E vs Fig 2C
and Fig 2D, bars 3 and 4) is perfectly consistent with the
fact that this precursor is transported into mammalian
microsomes both co- and post-translationally under
cotranslational conditions [29]
We reasoned that cross-linking of the ppl-86mer to
Sec61ap should also be detectable at the level of Sec61ap
and that quantification of cross-linking at the level of
Sec61ap should directly demonstrate the relevance of the
numbers stated above Single SRP–ribosome–nascent
pre-prolactin complexes were incubated with increasing
con-centrations of pancreatic microsomes Subsequently, the
microsomes were reisolated and subjected to chemical
cross-linking, or not cross-linked SDS/PAGE of the
samples, together with a serial dilutions of microsomes,
was followed by blotting to PVDF The blot was incubated
with anti-Sec61ap immunoglobulin and peroxidase
conju-gate of secondary antibodies The antibodies were
visual-ized by ECL of the blots and subsequent exposure to X-ray
film (Fig 3A,B) Indeed, an Sec61ap-related cross-linking
product was detected which comprised the radioactive
ppl-86mer (Fig 3C, lanes 6–9) This cross-linking product was specific as it depended on both transcript coding for ppl-86mer and cross-linking reagent and because it was not detected after puromycin chase and subsequent cross-linking (Fig 3D) Cross-cross-linking was quantified after den-sitometry of the X-ray films Between 27 and 35%, i.e around one out of three to four Sec61ap molecules could
be cross-linked to ppl-86mer under these conditions (Fig 3A,B, lanes 7 and 8) Thus, under conditions of saturation of microsomes with ppl-86mer, approximately every third or fourth Sec61ap molecule is in a position which allows cross-linking to p86mer and chase to pl-56mer, respectively According to these results, the con-centrations of specific binding sites for ribosome-nascent chain complexes of ppl-86mer in the microsomal suspen-sion are between 0.54 and 0.7 lM (average: 0.62 lM; Table 1) (note that the concentration of Sec61ap in the microsomal suspension is 2 lM; thus 27 and 35%, respectively, of cross-linked Sec61ap molecules correspond
to concentrations of productive binding sites of 0.54 and 0.7 lM; Table 1)
Content of canine pancreatic microsomes of proteins involved in protein transport and covalent modifications Cotranslational membrane association of nascent precur-sor proteins involves the SRP receptor (SR), comprising an a-subunit and a b-subunit Heterotrimeric Sec61p com-plexes form the core unit of the protein translocase In addition, protein translocase comprises the Hsp70 protein family members of the ER lumen (BiP/Grp78 and Grp170) and their putative membrane-integrated Hsp40 co-chaper-ones, Sec63p and Mtj1p Furthermore, Sec62p, TRAMp, and the TRAP complex appear to be additional subunits
of protein translocase As many precursor proteins that enter the ER are cotranslationally processed by the signal peptidase complex and the oligosaccharyl complex, these complexes must be in close proximity to protein translo-case Here we determined the concentration of these various components in the canine pancreatic microsomes, which had been used in the transport experiments discussed above, by semiquantitative immunoblot analysis (as des-cribed in the Experimental procedures) The results are summarized, together with some previously published data,
in Table 1
Discussion
Quantitative aspects of cell-free systems for the analysis
of protein transport into mammalian microsomes The transport data of this study suggest that the concen-tration of cotranslationally active protein translocases in defined suspensions of dog pancreas microsomes is
0.4–0.6 lM (average 0.5 lM) (Table 1) This agrees reasonably well with the previously observed concentration
of high-affinity ribosome-binding sites ( 0.3–0.4 lMat an Sec61ap concentration of 1.67 lM; Table 1) [6,8] Consid-ering that active protein translocase contains three to four heterotrimeric complexes [8,10–12], these functionally defined concentrations correspond to 1.5 or 2 lM hetero-trimeric Sec61p complexes, as present in cotranslationally
Trang 6active protein translocases Here we found that the
saturation of cotranslationally active protein translocases
with SRP–ribosome–nascent chain complexes inhibits
co-translational transport of other precursor polypeptides, but
allows post-translational transport This is consistent with
the numbers discussed above and the total concentration of
heterotrimeric Sec61p complexes (2 lM; Table 1) Recently,
we showed that co- and post-translational transport
involves the Sec61p complex [32] Thus, there are at least
two populations of Sec61p complexes present in pancreatic
microsomes; one class that provides the capacity for
cotranslational protein transport and one class that provides
the capacity for post-translational transport This is
some-what reminiscent of the situation in yeast [33] However, it
seems to us that in these mammalian microsomes the
concentration of SR, rather than the concentrations of the
translocase subunits Sec62p and Sec63p, may be the decisive
factor for the ratio between the two different populations of
Sec61p complexes (Table 1) We note that the concentration
of SRbp may be a more reliable indicator of the
concen-tration of SR because SRap has been shown to be rather
sensitive towards proteolytic attack during the isolation of
pancreatic microsomes
Typically, protein translocation is accompanied by
processing, by signal peptidase, of precursor proteins in
transit Furthermore, transient interaction of Sec61p
com-plexes with signal peptidase was observed during protein
translocation [21] Therefore, we argued that signal pepti-dase should be present in microsomes at a similar concen-tration as protein translocase Here we determined a concentration of 0.52 lM for signal peptidase (Table 1) Thus, it seems that a single signal peptidase complex is associated with protein translocase We take this as further substantiation of the concentrations discussed above and circumstantial evidence for the oligomeric character of protein translocase In contrast, most of the other subunits
of protein translocase, as well as the oligosaccharyl transf-erase complex, are present in pancreatic microsomes at similar concentrations as heterotrimeric Sec61p complexes Thus, multiple copies of these proteins and complexes may
be associated with oligomers of heterotrimeric Sec61p complexes in intact membranes of the mammalian ER
We note that our results are consistent with the idea that the protein translocase of the ER contains three or four heterotrimeric Sec61p complexes, but do not prove this However, it should be equally clear that the fact that the homologous complexes from bacteria or archaea, termed SecYEG [34] or SecY complex [35], respectively, were crystallized as dimers and monomers, does not necessarily mean that these complexes are active as monomers On the contrary, both electron microscopic [10–12] and, in particular, biophysical characterization [32,36] of active Sec61p complexes are consistent with an oligomeric state
Fig 3 Quantification of the association of Sec61ap with nascent
secretory proteins in single turnover experiments (A–C) Nascent
pre-prolactin (ppl-86mer) was synthesized in reticulocyte lysates (20 lL) in
the presence of [35S]methionine and dog pancreas microsomes at the
indicated concentrations [rough microsomes (RM), %, v/v] After
incubation for 20 min at 30 C, the translation reactions were
sub-jected to centrifugation (20 min, 15 000 g, 2 C) The pellets were
resuspended in buffer, divided into two aliquots, and incubated in the
absence (– XL) or presence (+ XL) of succinimidyl
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC, 335 l M ) for 20 min at
0 C The proteins were subjected to SDS/PAGE (15% acrylamide)
and subsequent blotting to poly(vinylidene difluoride) (PVDF)
mem-brane A threefold serial dilution series of microsomes was analyzed on
the same gel and blot (corresponding to 0.03, 0.1, 0.3, 1, and 3 lL of
RM; lanes 5 through 1) The blot was incubated with rabbit
anti-Sec61ap immunoglobulin and peroxidase conjugate of goat anti-rabbit
IgG The antibodies were visualized by ECL analysis of the blots and
subsequent exposure to X-ray film (15 and 30 s exposures are shown in
A and B, respectively) Subsequently, the blots were washed, dried and
subjected to autoradiography (C) (D) Nascent preprolactin
(ppl-86mer) was synthesized in rabbit reticulocyte lysate in the presence of
dog pancreas microsomes (7.5% RM, v/v) A mock translation minus
transcript was analyzed in parallel (– mRNA) After incubation for
20 min at 30 C, the translation reactions were centrifuged (20 min,
15 000 g, 2 C) The pellets were resuspended in buffer One aliquot
was incubated for 15 min at 30 C in the presence of puromycin
(+ puromycin) The aliquots were incubated in the absence (– XL) or
presence (+ XL) of SMCC (335 l M ) for 20 min at 0 C, as indicated.
The proteins were separated by SDS/PAGE (15% acrylamide) and
blotted to a PVDF membrane The blot was analyzed as described
above ppl 86 xSec61ap, ppl-86mer cross-linked to Sec61ap; ppl 86 ,
ppl-86mer.
Trang 7Quantitative considerations for the pancreatic ER
Typically, a microsomal preparation from a canine
pan-creas with an average weight of 40 g yields40 mL of the
defined microsomal suspension The concentration in
defined suspensions of dog pancreas microsomes, of 0.4–
0.6 lM, corresponds to a total of 20 nmol of
cotransla-tionally active protein translocases per canine pancreas
Thus, we calculate that 12· 1015 molecules of
cotransla-tionally active protein translocases are present per canine
pancreas, or about twice that number for a typical human
pancreas An average human produces 700 mL of
pancreatic juice per day The protein concentration of this
body fluid is 700 mgÆmL)1, thus the daily production of
secretory proteins in the human pancreas amounts to
5 g These 5 g correspond to 100 lmol, or 60 · 1018
molecules, of secretory proteins per day (average molecular
mass¼ 50 000 kDa) Therefore, one can estimate that
2500 molecules of presecretory proteins are handled per
cotranslationally active protein translocase in the human
pancreatic ER per day, or 100 molecules per hour
Assuming that the yield of microsomes in the course of a
microsomal preparation is never 100%, this number seems
at least to be in the correct order of magnitude when the
average speed of translation in a mammalian cell is
considered (up to 300 amino acid residues per min)
Acknowledgements
We wish to thank R Gilmore (Department of Biochemistry and
Molecular Biology, University of Massachusetts, Worchester, USA)
for a gift of anti-Ost48p serum This work was supported by the DFG
(grant C1/SFB 530) and by the Fonds der Chemischen Industrie.
References
1 Blobel, G & Dobberstein, B (1975) Transfer of proteins across
membranes I Presence of proteolytically processed and
unprocessed nascent immunoglobulin light chains on
membrane-bound ribosomes of murine myeloma J Cell Biol 67, 835–851.
2 Perara, E., Rothman, R.E & Lingappa, V.R (1986) Uncoupling
translocation from translation: implications for transport of
pro-teins across membranes Science 232, 348–352.
3 Walter, P & Blobel, G (1981) Translocation of proteins across the
endoplasmic reticulum II Signal recognition protein, SRP,
mediates the selective binding to microsomal membranes of
in-vitro-assembled polysomes synthesizing secretory protein.
J Cell Biol 91, 551–556.
4 Meyer, D.I., Krause, E & Dobberstein, B (1982) Secretory
pro-tein translocation across membranes – the role of the docking
protein Nature 297, 647–650.
5 Go¨rlich, D & Rapoport, T.A (1993) Protein translocation into
proteoliposomes reconstituted from purified components of the
endoplasmic reticulum membrane Cell 75, 615–630.
6 Kalies, K.-U., Go¨rlich, D & Rapoport, T.A (1994) Binding of
ribosomes to the rough endoplasmic reticulum mediated by the
Sec61p-complex J Cell Biol 126, 925–934.
7 Jungnickel, B & Rapoport, T.A (1995) A posttargeting signal
sequence recognition event in the endoplasmic reticulum
mem-brane Cell 82, 261–270.
8 Hanein, D., Matlack, K.E.S., Jungnickel, B., Plath, K., Kalies,
K.-U., Miller, K.R., Rapoport, T.A & Akey, C.W (1996)
Oligomeric rings of the Sec61p complex induced by ligands
required for protein translocation Cell 87, 721–732.
9 Tyedmers, J., Lerner, M., Bies, C., Dudek, J., Skowronek, M., Haas, I., Heim, N., Nastainczyk, W., Volkmer, J & Zimmer-mann, R (2000) Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes Proc Natl Acad Sci USA 97, 7214–7219.
10 Beckmann, R., Bubeck, D., Grassucci, R., Penczek, P., Verschoor, A., Blobel, G & Frank, J (1997) Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex Science
278, 2123–2126.
11 Beckmann, R., Spahn, C.M., Eswar, N., Helmers, J., Penczek, P.A., Sali, A., Frank, J & Blobel, G (2001) Architecture of the protein-conducting channel associated with the translating 80S ribosome Cell 107, 361–372.
12 Menetret, J.-F., Neuhof, A., Morgan, D.G., Plath, K., Rader-macher, M., Rapoport, T.A & Akey, C.W (2000) The structure
of ribosome-channel complexes engaged in protein translocation Mol Cell 6, 1219–1232.
13 Dierks, T., Volkmer, J., Schlenstedt, G., Jung, C., Sandholzer, U., Zachmann, K., Schlotterhose, P., Neifer, K., Schmidt, B & Zimmermann, R (1996) A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum EMBO J 15, 6932–6942.
14 Tyedmers, J., Lerner, M., Wiedmann, M., Volkmer, J & Zimmermann, R (2003) Polypeptide chain binding proteins medi-ate completion of cotranslational protein translocation into the mammalian endoplasmic reticulum EMBO Reports 4, 505–510.
15 Meyer, H.-A., Grau, H., Kraft, R., Kostka, S., Prehn, S., Kalies, K.-U & Hartmann, E (2000) Mammalian Sec61 is associated with Sec62 and Sec63 J Biol Chem 275, 14550–14557.
16 Dudek, J., Volkmer, J., Bies, C., Guth, S., Mu¨ller, A., Lerner, M., Feick, P., Scha¨fer, K.-H., Morgenstern, E., Hennessy, F., Blatch, G.L., Janoscheck, K., Heim, N., Frien, M., Nastainczyk, W & Zimmermann, R (2002) A novel type of cochaperone mediates transmembrane recruitment of DnaK-like chaperones to ribo-somes EMBO J 21, 2958–2967.
17 Voigt, S., Jungnickel, B., Hartmann, E & Rapoport, T.A (1996) Signal-sequence dependent function of the TRAM protein during early phases of protein transport across the ER membrane J Cell Biol 134, 25–35.
18 Fons, R.D., Bogert, B.A & Hegde, R.S (2003) Substrate-specific function of the translocon associated protein complex during translocation across the ER membrane J Cell Biol 160, 529–539.
19 Evans, E.A., Gilmore, R & Blobel, G (1986) Purification of microsomal signal peptidase as a complex Proc Natl Acad Sci USA 83, 581–585.
20 Kelleher, D.J., Kreibich, G & Gilmore, R (1992) Oligosacchary-ltransferase activity is associated with a protein complex com-posed of ribophorins I and II and a 48 kd protein Cell 69, 55–65.
21 Kalies, K.-U., Rapoport, T.A & Hartmann, E (1998) The
b subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translo-cation J Cell Biol 141, 887–894.
22 Scheper, W., Thaminy, S., Kais, S., Stagljar, I & Ro¨misch, K (2003) Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.
J Biol Chem 278, 37998–38003.
23 Wiertz, E.J.H., Tortoralla, D., Bogyo, M., Yu, J., Mothes, W., Jones, T.R., Rapoport, T.A & Ploegh, H.L (1996) Sec61-medi-ated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction Nature 384, 432–438.
24 Plemper, R.K., Bo¨mler, S., Bordallo, J., Sommer, T & Wolf, D.H (1997) Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation Nature 388, 891–895.
25 Tyedmers, J., Brunke, M., Lechte, M., Sandholzer, U., Dierks, T., Schlotterhose, P., Schmidt, B & Zimmermann, R (1996) Efficient folding of firefly luciferase after transport into mammalian
Trang 8microsomes in the absence of luminal chaperones and folding
catalysts J Biol Chem 271, 19509–19513.
26 Watts, C., Wickner, W & Zimmermann, R (1983) M13 procoat
and pre-immunoglobulin share processing specificity but use
dif-ferent membrane receptor mechanisms Proc Natl Acad Sci USA
80, 2809–2813.
27 Connolly, T & Gilmore, R (1986) Formation of a functional
ribosome-membrane junction during translocation requires the
participation of a GTP-binding protein J Cell Biol 103, 2253–
2261.
28 Murphy, E.C., III, Zheng, T & Nicchitta, C.V (1997)
Identifi-cation of a novel stage of ribosome/nascent chain association with
the endoplasmic reticulum membrane J Cell Biol 136, 1213–
1226.
29 Schlenstedt, G., Gudmundsson, G.H., Boman, H.G &
Zimmer-mann, R (1990) A large presecretory protein translocates both
cotranslationally, using signal recognition particle and ribosome,
and posttranslationally, without these ribonucleoparticles, when
synthesized in the presence of mammalian microsomes J Biol.
Chem 265, 13960–13968.
30 Bies, C., Guth, S., Janoschek, K., Nastainczyk, W., Volkmer, J.
& Zimmermann, R (1999) A Scj1p homolog and folding
catalysts present in dog pancreas microsomes Biol Chem 380, 1175–1182.
31 Hartmann, E., Wiedmann, M & Rapoport, T.A (1989) A membrane component of the endoplasmic reticulum that may be essential for protein translocation EMBO J 8, 2225–2229.
32 Wirth, A., Jung, M., Bies, C., Frien, M., Tyedmers, J., Zimmer-mann, R & Wagner, R (2003) The Sec61p complex is a dynamic precursor activated channel Mol Cell 12, 261–268.
33 Panzner, S., Dreier, L., Hartmann, E., Kostka, S & Rapoport, T.A (1995) Posttranslation protein transport in yeast recon-stituted with a purified complex of Sec proteins and Kar2p Cell
81, 561–570.
34 Breyton, C., Haase, W., Rapoport, T.A., Ku¨hlbrandt, W & Collinson, I (2002) Three-dimensional structure of the bacterial protein translocation complex, SecYEG Nature 418, 662–665.
35 van den Berg, B., Clemons, W.M., Collinson, I., Modis, Y., Hartmann, E., Harrison, S.C & Rapoport, T.A (2004) X-ray structure of a protein-conducting channel Nature 427, 36–44.
36 Hamman, B.D., Chen, J.C., Johnson, E.E & Johnson, A.E (1997) The aqueous pore through the translocon has a diameter of 40–60 A during co-translational protein translocation at the ER membrane Cell 89, 535–544.