Báo cáo khoa học: Protein transport into canine pancreatic microsomes A quantitative approach potx

Protein 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 o f p resecretory proteins i nto t he mammalian
rough e ndoplasmic reticulum involves a protein translocase
that comprises the integral membrane proteins Sec61ap,
Sec61bp, and Sec61cp as core components. Electron
microscopic analysis of protein tr anslocase i n r ough m icro-
somal membranes suggested that between three and four
heterotrimeric Sec61p complexes form th e central unit of
protein translocase. Here we analyzed the stoichiometry of
heterotrimeric Sec61p complexes p resent in cotranslationally
active protein translocases of canine p ancreatic 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 c apacity f or cotra nslational transport was estimated for
the e ndoplasmic reticulum of the human pancreas.
Keywords: endoplasmic reticulum; mammalian microsomes;
protein s ecretion; protein transport; pancreas.
Transport o f presecretory proteins into mammalian rough
microsomes involves cleavable signal peptides at the
N-terminus o f the p recursor proteins and a p rotein translo-
case in the microsomal m embrane [1]. Typically, transport
occurs as a sequence of three consecutive steps, namely (a)
specific membrane association of the precursor protein (also
termed t argetin g), ( b) m embran e inse rtion, a nd ( c) comple-
tion of translocation. S pecific membrane association 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]. P rotein t ranslocase (a) m ediates both
membrane insertion and completion of translocation, (b)
comprises Sec61ap, Sec61bp, and Sec61cp [5] as core
components, and ( c) operates c o- or post-translationally. I n
addition, heterotrimeric Sec61p complexes serve as specific
ribosome-binding sites in cotranslational transport [6], and
as sign al peptide receptors in g eneral [7]. The concentration
of heterotrimeric Sec61p complexes and specifically bound
ribosomes in defined s uspensions of mammalian microsomes
(absorbance ¼ 50 at 280 nm in 2 % SDS, c orresponding to 1
equivalent per lL) has b een determined as 1.67–2.12 l
M
[8,9]
and 0.27–0.39 l
M
[6,8], respective ly. These data were taken
as a first sugg estion that oligomers o f heterotrimeric S ec61p
complexes may be associated with ribosomes that are
simultaneously engaged in p rotein synthesis and transloca-
tion. Subse quently, cryo- and freeze fracture electron micro-
scopic analysis of the Sec61p complexes, as present i n 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 a nd facilitate insertion of presecretory protein s
into the Sec61p complex, as well as completion of translo-
cation [13,14]. These Hsp70 protein family members o f the
mammalian ER may be recruited t o 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 E R are processed by the signal peptidase c omplex [19]
and the oligosaccharyl transferase complex [20]. Therefore, it
is not s urprising that these complexes a re in close proximity
to protein translocase [21,22]. When misfolding occurs, the
polypeptides are exported to t he cytosol and degraded by the
proteasome. Protein export from the ER lumen to the cytosol
is also mediat ed 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 b e subunits of protein translocase
and involved in c ovalent 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:
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)
Eur. J. Biochem. 271, 3200–3207 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04252.x
type II and firefly luciferase were from Roche Diagnostics.
The peroxidase conjugate of goat antirabbit IgG was f rom
Sigma Chemical Company. [
35
S]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 ra bbit reticulocyte
lysates in the presence of [
35
S]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, C A, USA) usin g

IMAGEQUANT
software (version 5.1, Molecular Dynamics).
Alternatively, the proteins were t ransferred to P VDF
membranes a nd 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
PHOTOSHOP
software [version 3.0.5, Adobe Systems, Inc.,
San Jose, CA, USA] for production of figures. Luciferase
activity was determined as describe d previously [25].
Quantification of proteins synthesized
in 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 f olded 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 o f luciferase in subsequent experiments (data not
shown). In a second set of experiments, l uciferase was
synthesized in reticulocyte lysate in the presence of
[
35
S]methionine for 60 min. Subsequently, luciferase activity
and radioactivity, present in the luciferase b and after SDS/
PAGE, w ere 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 a nalyzed as described a bove. 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  2n
M
after in vitro translation f or 60 min, i.e. for the b atches used
of reticulocyte lysate and [
35
S]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 r espective 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 th at the concentration of a nascent p reprolactin
polypeptide chain (ppl-86mer, four methionines) and full-
length preprolactin (ppl, eight methionines) in the in vitro
system were  250 n
M
and 100 n
M
, 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 c omplex, s ignal
peptidase complex and o ligosaccharyl transferase complex
were purified according to previously published procedures
[5,17,18,20] and used for quantification according to our
previously published procedure [9]. B riefly, 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 o f 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 l ay 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 c aveat in mind.
Results
Capacity of canine pancreatic microsomes for
SRP-dependent protein transport
The ability to quantify n ewly synthesized proteins based on
radioactivity analyses and co mparison with simultaneously
synthesized firefly luciferase (as described in the Experi-
mental procedures) allowed us to analyze the e fficiency of
protein transport into canine pancreatic microsomes at
different ratios of the precursor a nd 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 tran slation reactions were divided into
four aliquots. One aliquot was untreated (Fig. 1A) and used
Ó FEBS 2004 Protein transport into mammalian microsomes (Eur. J. Biochem. 271) 3201
for determining the total quantity o f 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 t ranslation reaction (b) o f m icrosome-
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 t he quantities of Sec61p complexes [9] present in
the various translation r eactions. According to t he efficien-
cies of ppl-86mer synthesis, the ratios between precursor
and Sec61ap varied b etween 12.5 and 0.4 (Fig. 1 E, e.g. the
Table 1. Concentration of components in pancreatic rough microsomes
(RM). In the case of complexes, the prote ins, shown in pa rentheses,
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 eq uivalent per lL, or a protein concentration of
 15 mgÆmL
)1
or 0.33 m
M
(average molecular mass 50 000 kDa). BiP,
immunoglobulin heavy chain binding protein; OST, oligosaccharyl

transferase; SPase , signal peptida se; SRP, signal recognition particle.
Component Concentration Reference
High-affinity ribosome-binding sites
a
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]
TRAMp 1.74 l
M
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]
BiP 5.00 l
M
[30]
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
.
b
Productive 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 o f [
35
S]methionine and dog pancreas m icrosomes 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 w ere subjected to centrifugation (20 min, 15 0 00 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 resusp ended in buff er and incubate d
with 335 l
M
succinimidyl 4-(N-maleimidometh yl)cycloh exane -1-ca rb-
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 sample s (including th e luciferase
translation reaction) were subjectedtoSDS/PAGE(19.4%acrylamide
+ urea) and phosphorimager analysis. The q uantification of ppl-
86mer was carried out as described in the Experimental procedures
(E–H). The data for 1 lL(A)and5lL (B–D) aliquots are s hown. Th e
dotted line in (F) re presents th e sum of ppl-86m er a nd ppl-86mer cross-
linkedtoSec61ap 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
56
,pl-56mer.

3202 S. Guth et al. (Eur. J. Biochem. 271) Ó FEBS 2004
first 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 t o p l-56mer, increased with increasing concentration
of microsomes. A s e xpected, at t he low est concentration of
microsomes, the total binding exceeded by far the specific
binding [28] (Fig. 1F vs. 1G). However, the specific bindin g
was significant at higher concentrations of microsomes
(Fig. 1 F vs. 1G). There was a g ood corre lation 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. 1 G 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 F ig. 1, indicate concentrations of specific binding
sites for ribosome–nascent chain complexes o f ppl-86mer in
the microsomal suspen sion 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 l
M
(Fig. 1 G, e.g. the
third data point/160 nmol Sec61ap: 38 nmol ppl-
86mer · 12.5 ¼ 475 nmol) (average: 0.4 l
M
;Table 1)(note
that the c oncentration of S ec61ap in the microsomal
suspension is  2 l
M

; Table 1). As cross-linking typically
does not occur at efficiencies of 100%, the latter number
seems to be more reliable. Thus, at t hese intermediate
concentrations of microsomes, the average ratio between
specifically bound ppl-86mer and Sec61 ap was 1.0 : 4.3
(Fig. 1 G; 0.46 /2 l
M
corresponds 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 we re 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 t o c onfirm 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
[
35
S]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 [
35
S]methionine, i.e. with or without prior
EDTA treatment. In parallel, microsomes were subjected to
a first mock tr anslation reaction in the absence of transcript

and [
35
S]methionine and, after reisolation and treatment
with or without EDTA, t o a s econd translation reaction i n
the p resence of [
35
S]methionine. T he precursor preprolactin
was synthesized in the second stage of the experiment, and
the various microsomes were present in t hese 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 nas cent presecretory p roteins to microsomes
in a single turnover experiment prevents cotranslational transport of
other presecretory proteins i n a s ub sequen t transport reaction. Nascent
preprolactin (p pl-86mer) was synthesizedinreticulocytelysateinthe
presence o f 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 m in at 30 °C, the
translation r eactions 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 m in a t 3 0 °C. MgCl
2
(7.5 m
M
)wasaddedtoallsampleswhichwere
then adjusted to the same final concentration of EDTA. Subsequently,
preprolactin w as synthesized in reticulocyte lysates in the presence of

[
35
S]methionine and in the simultaneous pre sence 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 p resence of [
35
S]methionine and in
the simultan eous p resence o f dog pancreas micros omes at the indicated
concentrations (RM, %, v/v) (B). After incubation for 45 min at
30 °C, the t ranslation reactions were s upplemented 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 p pl-86mer was synthesized in the
second translation rea ction 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.
Ó FEBS 2004 Protein transport into mammalian microsomes (Eur. J. Biochem. 271) 3203
translation confirmed that the concentrations of micro-
somes that were used in t he secon d stage of the two-stage
experiment allowed the detectio n of quantitative differences
in transport e fficiencies (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, lan es/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, transpo rt o f 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. 2 A,C, lanes/bars 1 and 2). Furthermore, microsomes
were minimally affected by the first mock translation
(Fig. 2 A,C, lanes/bars 5–8). Thus, the two-stage experiment
demonstrated that the single turnover e xperiments had led
to saturation of microsomes with respect to th eir 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 t ranspor-
ted into mammalian microsomes only cotranslationally and
that the preprocecropin–dihydrofolate r eductase hybrid
is transported into mammalian microsomes both co-
and post-translationally under c otranslational conditions
(Fig. 2 E) and, obviously, only post-translationally under

post-translational conditions (Fig. 2F) [29]. SRP-independ-
ent a nd post-tr anslational t ransport of the preprocecropin–
dihydrofolate reductase hybrid was not affected by satura-
tion of microsomes with respect to their cotranslational
transport capacity (Fig. 2F). We note that the observation
that the preprocecropin–dihydrofolate r eductase 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 s hould 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 P VDF. The b lot was i ncubated
with anti-Sec61ap immunoglobulin and peroxidase conju-
gate of secondary antibodies. The antibodies were visual-
ized by ECL of the blots and subseq uent 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-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. 3 A,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 ppl-86mer 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 l
M
(average: 0.62 l
M
;
Table 1 ) (note that the concentration of Sec61apinthe
microsomal suspension is  2 l
M
; t hus 27 and 35%,
respectively, of cross-linked Sec61ap molecules correspond
to concentrations of productive binding sites o f 0.54 and
0.7 l
M
;Table1).
Content of canine pancreatic microsomes of proteins
involved in protein transport and covalent modifications
Cotranslational membrane association of nascent precur-

sor p roteins involves the SRP receptor (SR), comprising an
a-subun it 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 member s of the ER lumen (BiP/Grp78 a nd Grp170)
and their putative m embrane-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 t he signal
peptidase complex and the oligosaccharyl complex, these
complexes must b e in close proximity to pro tein translo-
case. Here w e d etermined t he 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 d es-
cribed in the Experimental procedures). The r esults are
summarized, together w ith some p reviously 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 l
M
(average 0.5 l
M
) (Table 1). This agrees

reasonably well with the previously observed concentration
of high-affinity ribosome-binding sites ( 0.3–0.4 l
M
at an
Sec61apconcentrationof1.67l
M
; Table 1) [6,8]. Consid-
ering that active protein translocase contains three to fou r
heterotrimeric complexes [8,10–12], these functionally
defined concentrations correspond to 1.5 or 2 l
M
hetero-
trimeric Sec61p complexes, as present in cotranslationally
3204 S. Guth et al. (Eur. J. Biochem. 271) Ó FEBS 2004
active protein translocases. Here we found that the
saturation of cotranslationally active protein translocases
with SRP–ribosome–nascent chain complexes inhibits co-
translational t ransport o f other precursor polypeptides, but
allows post-translational transport. This is consistent with
the numbers discussed above and the t otal concentration of
heterotrimeric Sec61p complexes ( 2 l
M
; 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 p resent in pancreatic
microsomes; one class that provides the capacity for
cotranslational protein transport and one class that provides
the c apacity for post-translational transport. This is some-
what reminiscent of t he 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 t owards 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 i nteraction 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 a s protein tra nslocase. Here we de termined a
concentration of 0.52 l
M
for signal peptidase (Table 1).
Thus, it seems that a single signal peptidase complex is
associated with protein t ranslocase. We take this as further
substantiation of the concentrations discussed above and
circumstantial evidence for the oligomeric character of
protein translocase. In c ontrast, most of the other subunits
of protein translocase, as well as the oligosaccharyl transf-
erase complex, are present in p ancreatic microsomes a t
similar concentrations as heterotrimeric Sec 61p complexes.
Thus, multiple copies of these proteins and complexes may
be associated with oligomers o f 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 cont ains t hree o r
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, doe s not
necessarily mean that these complexes are active as
monomers. On the contrary, both electron microscopic
[10–12] a nd, 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 pre-
secretory proteins in single turnover experiments. (A–C) Nascent pre-
prolactin (ppl-86mer) was synthesized in reticulocyte lysates (20 lL) in
the presence o f [
35
S]methionine and dog pancreas microsomes at the
indicated concentrations [rough microsom es (RM), % , v/v]. A fter
incubation fo r 2 0 m in 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
)for20minat
0 °C. The proteins were subjected to SDS/PAGE (15% acrylamide)
and subsequent blo tting to p oly(vinylide ne difluoride) (PVDF) mem -
brane. A threefold serial dilution series of microsomes was analyzed on
the same g el and blot (corresponding to 0.03, 0 .1, 0.3, 1 , and 3 lLof

RM; lanes 5 through 1). The blot was incubated with rabbit anti-
Sec61ap immunoglobulin and peroxidase c onjugate of goat anti-rabbit
IgG. The antibodies were visualized by EC L analysis of the blots and
subsequent exposure t o 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 synthesiz ed in rabbit reticulocyte lysate in the presence of
dog pancreas microsome s (7.5% RM , v/v). A mock translation minus
transcript was analyzed in parallel (– mRNA). After incubation for
20 m in at 30 °C, the t ranslation reactions were centrifuged (20 min,
15 000 g,2°C). The pellets were resuspen ded in b uffer. One aliquo t
was incubated for 15 min a t 30 °C in the presen ce of puromycin
(+ puromycin). The aliquots were incubated in the absence (– XL ) or
presence (+ X L) of SMCC (335 l
M
)for20minat0°C, as indicated.
The prote ins were separated by SDS/PAGE (1 5% acrylamide) an d
blotted to a PVDF membrane. The blot was analyzed as de scribed
above. pp l
86
xSec61ap, pp l-86mer cross-linked to Sec61 ap; ppl
86
,
ppl-86mer.
Ó FEBS 2004 Protein transport into mammalian microsomes (Eur. J. Biochem. 271) 3205
Quantitative considerations for the pancreatic ER
Typically, a microsomal preparation from a canine pan-
creas with an average weight of 40 g yields 40 mL of the
defined microsomal suspension. The concentration in
defined suspensions of dog pancreas microsomes, of 0.4–

0.6 l
M
, corresponds to a total of 20 nmol of cotransla-
tionally active protein translocases p er canine pancreas.
Thus, we calculate that 12 · 10
15
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  70 0 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 · 10
18
molecules, of secretory proteins per day (average molecular
mass ¼ 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 mamm alian cell is
considered (up t o 300 amino acid residues per min).
Acknowledgements
We wish to thank R. Gilmore (DepartmentofBiochemistryand
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 Che mischen Industrie.
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