(Received for publication, April 18, 1995; and in revised form, June 9, 1995)
From the
The present study was undertaken to investigate the
Ca binding properties of sarcoplasmic reticulum
Ca
-ATPase after removal of the cytoplasmic regions by
treatment with proteinase K. One of the proteolysis cleavage sites (at
the end of M6) was found unexpectedly close to the predicted
membrane-water interphase, but otherwise the cleavage pattern was
consistent with the presence of 10 transmembrane ATPase segments.
C-terminal membranous peptides containing the putative transmembrane
segments M7 to M10 accumulated after prolonged proteolysis, as well as
large water-soluble fragments containing most of the phosphorylation
and ATP-binding domain. Ca
binding was intact after
cleavage of the polypeptide chain in the N-terminal region, but cuts at
other locations disrupted the high affinity binding and sequential
dissociation properties characteristic of native sarcoplasmic
reticulum, leaving the translocation sites with only weak affinity for
Ca
. High affinity Ca
binding could
only be maintained when proteolysis and subsequent manipulations took
place in the presence of a Ca
concentration high
enough to ensure permanent occupation of the binding sites with
Ca
. We conclude that in the absence of
Ca
, the complex of membrane-spanning segments in
proteolyzed Ca
-ATPase is labile, probably because of
relatively free movement or rearrangement of individual segments. Our
study, which is discussed in relation to results obtained on
Na
,K
-ATPase and
H
,K
-ATPase, emphasizes the importance
of the cytosolic segments of the main polypeptide chain in exerting
constraints on the intramembranous domain of a P-type ATPase.
The way in which transmembrane segments of a membrane protein interact to form organized intramembranous domains with functional properties is still a matter of speculation. There is evidence that during the initial stages of protein biosynthesis, insertion into the membrane is governed by local factors in the polypeptide chain such as the presence of hydrophobic segments and neighboring charged amino acid residues, which can function as transfer and stop signals for insertion of the nascent polypeptide chain into the lipid bilayer (Friedlander and Blobel, 1985; Audiger et al., 1987; Lipp et al., 1989; Boyd and Beckwith, 1990; Dalbey, 1990; Sipos and von Heijne, 1993; Anderson and von Heijne, 1994). According to the two-stage hypothesis, as initially proposed by Popot and Engelman (1990), preformed transmembranes helices with independent stability would then pack into the final stable and compact tertiary fold with functional properties. The hypothesis that transmembrane helices behave as autonomous folding domains and acquire most of their native structure independently of the rest of the molecule has prompted a large number of studies. Many laboratories have attempted to reconstitute functional membrane proteins like receptors, bacteriorhodopsin, sugar transporters, or P-glycoprotein, from complementary fragments generally obtained by proteolysis or by co-expression in a host membrane (e.g. Kobilka et al.(1988), Kahn and Engelman(1992), Bibi and Kaback(1992), Popot(1993), Lemmon and Engelman(1994), Cope et al. (1994), and Loo and Clarke(1994)). Judging from published reports, these efforts have up to now been reasonably successful, at least when starting from a small number of large complementary fragments, generally including the extramembranous domains.
In relation to the above mentioned data, it is of interest
to know whether autonomous folding applies to all kinds of integral
membrane proteins or whether also interaction with cytoplasmic domains
is decisive for stability. In this connection it should be noted that
several of the proteins previously subjected to study are characterized
by a predominance of membrane-embedded segments. In this respect,
P-type ion pumps are of particular interest because of the presence of
both large cytosolic domains and transmembrane domains that must
accommodate the hydrophilic pathways for ion translocation. One example
is provided by Na,K
-ATPase, involved
in the Na
/K
exchange across the
plasma membrane in animal cells. Maturation of newly synthesized
Na
,K
-ATPase, with acquisition of
cation transporting properties, occurs after formation of a
heterodimeric complex with the glycosylated
-subunit and export to
the plasma membrane (Geering 1991; Fambrough et al., 1994;
Schmalzing and Gloor 1994). It has been demonstrated that with mature
Na
,K
-ATPase as well as with the
related H
,K
-ATPase, it is possible
after proteolytic removal of the cytosolic domains to retain an
intramembranous complex with an intact ability to occlude K
(or Rb
) and Na
; such
preparations are often referred to as ``19-kDa'' membranes
because they are formed by noncovalent interaction between the
remaining separated membrane polypeptide fragments, including a large
C-terminal proteolytic fragment of approximately this size (Karlish et al., 1990; Esmann and Sottrup-Jensen, 1992; Rabon et
al., 1993). However, reports with
Na
,K
-ATPase 19-kDa membranes suggest
that these membranous fragments have an increased susceptibility to
thermal inactivation (Or et al., 1993: Shainskaya and Karlish,
1994).
In the closely related SERCA ATPases, Ca transport does not require the presence of a
-subunit, and
the ATPase chain is retained in the SR(
)/ER membrane after
biosynthesis, presumably without undergoing any drastic
posttranslational modification other than formation of a few disulfide
bonds. In a previous study on the transmembrane organization of the
SERCA1a Ca
-ATPase polypeptide chain, an ATPase
fragment of approximately 30 kDa, relatively resistant to proteolysis,
was reported to be formed by incubation with proteinase K (Matthews et al., 1990). Based on the immunoreactivity of this fragment,
it was thought to correspond to the whole C-terminal
membrane-associated domain of the ATPase. In addition, Matthews et
al.(1990) indicated the presence of a smaller fragment (about 19
kDa) with the same immunoreactivity as the 30-kDa peptide, presumably
analogous to the C-terminal fragment produced by proteolytic treatment
of Na
,K
-ATPase.
In
Ca-ATPase as in
Na
,K
-ATPase, residues critical for
cation binding or occlusion are believed to be restricted to the
protein transmembrane domain (Clarke et al., 1989; le Maire et al., 1990; Sumbilla et al., 1991; Vilsen and
Andersen, 1992; Jewell-Motz and Lingrel, 1993; Vilsen, 1993; Skerjanc et al., 1993; Toyoshima et al., 1993; Andersen, 1994;
Andersen and Vilsen, 1994, 1995). Thus, the way seemed open to test if
Ca
-ATPase, with a simpler structure than
Na
,K
-ATPase, would also be able to
retain cation binding after removal of the cytoplasmic domains by
proteolytic cleavage. To do so, we first analyzed in detail the
cleavage pattern of SR Ca
-ATPase with proteinase K.
We then proceeded to test how the Ca
-binding
properties correlated with the extent of proteolytic cleavage at
various stages of proteolysis. Our results clearly show that even
relatively large transmembrane fragments of Ca
-ATPase
rapidly experience loss of the ability to control the permeation
pathway and to retain high affinity Ca
binding,
unless a very high Ca
concentration keeps the
Ca
binding cleft permanently occupied during
proteolysis. These results provide support for the concept that for
P-type ATPases, interaction with stabilizing extramembranous segments,
as well as cation binding to the intramembranous sites, is crucial for
maintenance of a native-like structure of the transmembrane portion of
the protein.
Figure 5:
Effect of proteinase K treatment on Ca
binding and
Ca
-dependent phosphorylation from
[
-
P]ATP. A, after incubation of SR
vesicles with proteinase K for various periods in EGTA/Mg medium (see
``Experimental Procedures''), we measured their ability to
bind
Ca
(circles) under our
standard conditions (buffer A, at pH 7.5, see ``Experimental
Procedures''; the protocol was similar to that of the experiment
shown in Fig. 4A), as well as the amount of
phosphoenzyme (triangles) formed from 10 µM
[
-
P]ATP in buffer A plus 0.2 mM Ca
, at 4 °C (see ``Experimental
Procedures''). PanelsB and C, SR
vesicles were incubated with proteinase K for 15 min in either 100
µM Ca
medium (lanes1 and 2) or EGTA medium (without Mg
, lanes3 and 4) or left intact (lanes5 and 6) and were then incubated with
[
-
P]ATP either in the presence of
Ca
(lanes with oddnumbers) or in the presence of excess EGTA (laneswith evennumbers) before being
quenched with acid and centrifuged. Resuspended samples were run on a
Laemmli gel containing 6.5% acrylamide to enhance resolution between
the large fragments, and the gel was both autoradiographed with a
PhosphorImager (C) and stained with Coomassie Blue (B).
Figure 1:
Time dependence of proteolytic attack
by proteinase K in the presence of micromolar Ca and
the effect of Ca
removal. Left, for SDS-PAGE
on a Laemmli gel, lanes were all loaded with 20 µg of total
protein: either native SR vesicles (lane9) or SR
vesicles incubated with proteinase K for various periods (as indicated)
in the presence of 100 µM Ca
(see
``Experimental Procedures''). Time zero (lane2) corresponds to a sample for which proteinase K had
been inhibited with PMSF before SR was added. Lane1 contains molecular mass markers (LMW Pharmacia kit), one of which
(
-lactalbumin) is a 14.4-kDa protein with abnormal migration
behavior; it is running faster than lysozyme, although both proteins
have identical molecular mass (see Fig. 1of le Maire et
al., 1993), and also faster than a 14.4-kDa CNBr fragment of
myoglobin (data not shown). As a result,
-lactalbumin migrates
with an apparent molecular mass close to 12 kDa. Right, a
different but similar experiment, in which SR proteolysis took place
during 15 min either in the Ca
-containing medium (lane10) or in an EGTA- and
Mg
-containing medium (lane11).
Figure 7:
Effect
of a high Mg or Ca
concentration
during proteolysis on the Ca
sensitivity of intrinsic
fluorescence after proteolysis (A) and the proteolysis pattern (B). The 100 mM bis-Tris proteolysis medium (pH 6.5)
contained either a, 0.3 mM Ca
(left); b, 0.3 mM Ca
and 10 mM Mg
(center); or c, 10 mM Ca
alone (right). A, after the indicated periods, as for the experiment
illustrated in Fig. 6A, proteolyzed samples were
diluted 20-fold into buffer A and intrinsic fluorescence was recorded.
EGTA was first added (closedarrowheads; the EGTA
concentration was a, 100 µM; b, 100
µM; or c, 2 mM, since the initial
Ca
concentration was 0.5 mM in this case),
followed by Ca
(doubleopenarrowheads; the total concentration of Ca
was a, 1, mM; b, 1
mM; or c, 3 mM). B, proteolysis,
performed under the same conditions as above, was arrested with PMSF
after 5, 15, 30, or 80 min, and the samples were run on a Tricine gel.
The lastlane shows molecular mass standards. Note
that p19 migrates at approximately the same rate as myoglobin (17 kDa
marker) in this gel system.
Figure 2:
Characterization of
Ca-ATPase fragments after proteolytic attack by
proteinase K from their sedimentability (A) and
immunoreactivity (B and C). For panelA experiments, SR vesicles were first incubated with proteinase K
for 15 min in 100 µM Ca
medium. Then,
part of the sample was centrifuged at high speed, and the pelletable (Mb.) and soluble (Cyt.) fractions were separated by
SDS-PAGE on a Laemmli gel together with an aliquot of the total sample (Tot.) and molecular mass standards (LMW). The gel
was stained with Coomassie Blue. For panelB experiment, Ca
-ATPase fragments (total sample)
obtained after 5 min of proteinase K treatment were separated by
SDS-PAGE, again on a Laemmli gel, and blotted onto a PVDF membrane. In
one case (lane1), the blot was incubated with Ab
877-888 (an antibody raised against residues 877-888 in the
C-terminal part of the ATPase); the asterisk indicates the
band referred to as p20. In the other case (lane2),
the blot was incubated with Ab 1-12, raised against the very
first N-terminal residues of the ATPase. In both cases, bound antibody
was revealed first (lanes1a and 2a), and
the blots were subsequently stained with Coomassie Blue (lanes1b and 2b, respectively). PanelC experiment was similar to the one illustrated in B,
except that 16% gels, prepared according to
Schägger and von Jagow(1987), were used. After 10 (lanes1 and 4) or 15 min (lanes2 and 3) proteolysis in the presence of 100
µM Ca
, Ca
-ATPase
fragments, were separated by electrophoresis, blotted onto PVDF, and
first revealed by an antibody, either Ab 78(7) (lane2a), which reacts with the N-terminal region ATPase (see
``Experimental Procedures''), or Ab 577-588 (lane3a), which reacts with the central region, or Ab
796-806 (lane4a), which reacts with the
C-terminal region. The Western blots were subsequently stained with
Coomassie Blue (lanes2b, 3b, and 4b, respectively). Lane1 on the left shows Coomassie Blue staining of the fragments on the gel itself,
corrected for gel shrinkage. Note that in Tricine gels, proteinase K
migrates at the same speed as p30, so that it cannot be seen in this
figure (but see lane1 in Fig. 7B).
In addition, in these gels, p54 migrates at a speed intermediate
between those of M55 and calsequestrin, the latter protein being the
fastest; note also that calsequestrin transfer onto PVDF is not
efficient, compared with transfer of other proteins (compare lane1 and lanes2b, 3b, or 4b). The approximate location of molecular mass markers is
indicated on the right (LMW).
Figure 4:
Loss of Ca
binding and sequential dissociation properties of SR vesicles
after proteolysis attack in the presence of 100 µM Ca
, and parallel quantification of the
concomitant appearance of the main Coomassie Blue-stained fragments.
For panelsA and B, SR vesicles were
incubated with proteinase K in 100 µM Ca
medium for various periods before proteolysis arrest by PMSF. A, aliquots (100 µg of protein) were then diluted in a pH
7.5 buffer (buffer A, see ``Experimental Procedures''),
adsorbed on a nitrocellulose membrane, and perfused with the same
buffer supplemented with
Ca
(and
[
H]glucose) at pCa 5 so that equilibrium
Ca
binding to the adsorbed membranes was
measured (circles). The results shown here are representative
of several experiments (performed in triplicate) leading to the same
results. Identical
Ca
-equilibrated
samples were also prepared for subsequent perfusion with 2 ml of the
same medium with no
Ca
, but either 1
mM nonradioactive
Ca
or 2
mM EGTA (this second perfusion lasted 2 s), and residual
Ca
was measured (squares and triangles, respectively). For intact SR vesicles, the complete
time dependence of the result of this second perfusion is illustrated
in panelC. Here, this perfusion was performed with a
Biologic rapid filtration apparatus for various periods; the perfusion
medium again contained either 1 mM
Ca
(squares) or 2 mM EGTA (triangles).
Interpretation for this experiment is shown in D, with
Ca
being represented by closedcircles and
Ca
by opencircles. B, proteolyzed samples, similar to
those used for the
Ca
measurements, were
run on an SDS gel (cf. Fig. 1) and the main Coomassie
Blue-stained bands were scanned and quantified. The ordinateaxis represents integration of the optical density of the
main bands: intact ATPase (triangles), p81/83 (circles), p29/30 (diamonds), p27/28 (squares), and p19 (asterisks).
Figure 6:
Intrinsic fluorescence level and
Ca-induced fluorescence changes of SR vesicles after
proteolysis. A, SR proteolysis took place in 100 µM calcium medium. At various times before (t = 0) or
after proteinase K addition, as indicated, 100-µl aliquots of
proteolyzed SR were diluted in 1.9 ml of buffer A, supplemented with 10
µM Ca
, and intrinsic fluorescence was
recorded. The final concentration of Ca
after SR
addition was close to 20 µM, taking contaminating
Ca
into consideration. 100 µM EGTA was
subsequently added (filledarrowhead), followed by 90
µM Ca
(final pCa close to 5, singleopenarrowhead) and later on by 1
mM Ca
(doublearrowhead).
Each addition was performed with 2 µl of concentrated solution,
corresponding to a very small dilution that was corrected for (0.1%). PanelsB and C, two batches of SR vesicles
were prepared, corresponding to either 30 min proteolysis followed by
PMSF quench (C) or to no proteolysis (in this case, proteinase
K was quenched with PMSF before addition of SR, panelB). 100 µl of membrane suspension was diluted into
1.9 ml of buffer A, supplemented with 20 µM Ca
(final pCa was therefore close to 4.5). Then, starting
from this fluorescence level (corresponding to the closed symbols at pCa 4.5), we measured the relative changes in intrinsic
fluorescence (expressed as percent of the initial fluorescence level)
observed when EGTA or MgEDTA (de Foresta et al., 1994) was
added to reduce the free Ca
concentration or when
Ca
was added to increase
it.
In some cases, Ca
binding was also examined by gel
equilibration chromatography (Inesi et al., 1980) after
separation of soluble fragments and proteinase K from the membranes by
ultracentrifugation. The pelleted membranes were resuspended in a pH
7.5 buffer containing 100 mM KCl and 10 mM Tes-Tris,
to which 55 µM
Ca
(740
kBq/liter) plus 40 µM EGTA were added. This sample was
applied to a column (1
30 cm of Sephadex G-50, Pharmacia
Biotech Inc.), which had been preequilibrated with the same buffer.
Ca
binding was calculated from the rise in
Ca
in the eluted fractions. The results
obtained with both techniques for
Ca
binding measurement were quantitatively similar, both before and
after proteinase K treatment.
Figure 3:
Schematic representation of the location
of various cleavage sites, relative to the predicted topology of ATPase (A), and linear map of the various fragments identified after
proteolytic attack by proteinase K (B). A, schematic
view of Ca-ATPase predicted folding (numbers for the
residues predicted to lie at each membrane/water interface are taken
from Clarke et al. (1990); the dashedhorizontalline schematizes the position of the top of the stalk)
with the position of the main sites for proteinase K attack. The
indicated numbers correspond to the residues immediately following the
cleavage sites (120 refers to the Leu-119-Lys-120
peptide bond, 243 refers to the Thr-242-Glu-243 peptide
bond). Sites for V8 cleavage (V8
at Glu-231-Ile-232
and V8
at Glu-715-Ile-716) and tryptic cleavage
(T
at Arg-505-Ala-506 and T
at
Arg-198-Ala-199) are also indicated, as well as the
phosphorylation site (P) at Asp-351. Openboxes in the membranous domain correspond to critical residues, Glu-309,
Glu-771, Asn-796, Thr-799, Asp-800, and Glu-908 (Clarke et
al., 1989), the latter being controversial (Vilsen and Andersen,
1992). The approximate locations of the epitopes for our antibodies
(1-12; 403-417; 577-588; 796-806;
877-888; 985-994) are indicated by filledboxes. Questionmarks indicate cleavage
sites not precisely determined. B, linear map for the main
Ca
-ATPase fragments identified after proteinase K
treatment.
Bands of lower molecular mass (less than 14 kDa) also appear in significant amount as a result of proteolysis (data not shown). Bands representing components with apparent molecular masses about 43 and 37/38 kDa are also visible on Fig. 1after proteolysis. Finally, the bands in the 55-60 kDa region in Fig. 1, visible both in native SR and after proteolysis, correspond to calsequestrin and M55 glycoprotein, as documented previously (le Maire et al., 1990).
When similar proteolysis experiments were
repeated in the presence of EGTA together with Mg and
Na
(to minimize the EGTA-induced instability of
Ca
-ATPase), the rate of proteolytic degradation was
slowed down; otherwise, we observed essentially the same pattern of
proteolytic degradation as in the presence of Ca
,
except that a new protein component (p95) became prominent (see lane11 in Fig. 1). Binding of Ca
to sarcoplasmic reticulum Ca
-ATPase was
previously found to greatly affect its sensitivity to V8 protease also,
especially at Glu-231-Ile-232 (le Maire et al., 1990),
as well as the sensitivity to trypsin of the peptidic bond located
after Arg-198 (Andersen et al., 1986). Thus, it appears that
the rate of proteolysis attack at various sites is modified by
Ca
, but, as in the case of V8, the same peptides are
probably formed, as shown by the similar mobility of the bands formed
in the presence or absence of Ca
(see lanes10 and 11 in Fig. 1).
We then focused on the region corresponding to molecular masses around 20-30 kDa, which were reported by Matthews et al.(1990) to contain the C-terminal membranous fragments. In the experiment illustrated in Fig. 2A, SR vesicles were first incubated with proteinase K for 15 min. An aliquot of the total sample was kept for electrophoretic analysis (Tot.), while another aliquot was submitted to high speed centrifugation to separate membrane-bound (Mb.) and cytosolic soluble (Cyt.) fragments. To our surprise, a vast majority of the p29/30 component accumulating after long proteolysis periods (Fig. 1) was found in the soluble fraction, excluding that it could represent the fragment corresponding to the C-terminal transmembrane segments of the ATPase. The supernatant was extraordinarily enriched in this p29/30 peptide, whereas, besides proteinase K, the only other peptide of significant length present was p14. All the other proteolytic peptides, including p27/28, p19, and part of p14, were recovered in the pelletable fraction (Fig. 2A, lane3). This was also the case for the intravesicular proteins calsequestrin and M55, suggesting that the vesicles remained sealed after treatment with proteinase K.
The bands were further characterized after transfer to PVDF
membranes and immunodetection with various antibodies. PanelB shows two blots, incubated with an antibody directed
against peptide 877-888 (lane1a) and with an
antibody directed against peptide 1-12 (lane2a), respectively. The same blots were subsequently
stained with Coomassie Blue (lanes1b and 2b, respectively). The C-terminal antibody (lane1a) reacted with p28, p27, and p19 as well as with a
fainter band referred to as p20 (see asterisk), slightly
larger than p19 and hardly visible in the Coomassie Blue-stained gel
under these conditions. Similar results were obtained using an antibody
raised against the very last residues of the ATPase, namely against
peptide 985-994 (data not shown). The N-terminal antibody (lane2a) reacted strongly with the p28 region, as
well as with a 14-kDa component. In addition, both antibodies reacted
with undegraded ATPase and p81/83, but not with p29/30 (see below).
Results similar to those observed with the antibody against peptide
1-12 were obtained using the polyclonal antibody 78(7), whose
main epitope is located in the N-terminal one-fifth of the ATPase (see
``Experimental Procedures''). In this case, Ab 78(7)
decorated the lower edge of the 81/83 band, indicating the presence of
two components in this region; these two bands could be clearly
differentiated when a Laemmli gel with a lower acrylamide content was
used (e.g. see Fig. 5B, below). To be able to
resolve the 27/28-kDa components, it turned out to be important to use,
instead of the Laemmli system, the Tricine gel system devised by
Schägger and von Jagow(1987). The latter gel system
resulted in a higher relative mobility for the C-terminal fragments and
thus allowed us to differentiate the N- and C-terminal components of
p28 (Fig. 2C, lanes2a and 4a). Coomassie Blue staining intensities, in turn (Fig. 2C, lane1), led to the
conclusion that the major part of p28 is represented by the N-terminal
fragment. In the same series of experiments, the p29/30 fragments were
clearly stained by an antibody against peptide 577-588 in the
central cytosolic domain of Ca-ATPase (Fig. 2C, lane3a), as expected from
the fact that these fragments were recovered in the supernatant (panelA). Thus, after proteolysis in our standard
100 µM Ca
medium, the major peptides
products present in the 27-30 kDa region originate from the
N-terminal and central regions, whereas peptides originating from the
C-terminal region are mainly present as smaller 19-kDa fragments, as
was previously found for Na
,K
-ATPase
and H
,K
-ATPase.
Fig. 3illustrates the location of all of
these proteolysis sites in the Ca-ATPase sequence.
The formation of primary and secondary proteolytic degradation products
can be accounted for by the presence of seven characteristic main
proteolytic regions, five of which are unambiguously localized: 1) the
Leu-119-Lys-120 peptide bond located after M2 in the S2 region,
2) the Thr-242-Glu-243 peptide bond at the beginning of the S3
region, 3) the Cys-349-Ser-350 and Leu-356-Thr-357 peptide
bonds immediately before and after the phosphorylation site (Asp-351)
(cleavage at Thr-345-Ser-346 and Asn-359-Gln-360 was also
detected, in low amounts), 4) various peptide bonds in the
733-747 region, preceding S5/M5, and 5) the Met-817-Asp-818
peptide bond between M6 and M7, as well as the nearby
Leu-807-Gly-808 bond. Furthermore, based on ESI-MS, p29/30
presumably ends at Ser-610-Ile-611 (see above). Since by
N-terminal sequencing we did not detect any peptide starting at this
position and ending at 733-747 (the molecular mass of such
peptide would be close to 14 kDa), additional cleavage sites must be
present in the intervening sequence (comprising the ``hinge''
region), resulting in the formation of small peptides not detected by
gel electrophoresis and probably released into the supernatant. The
observed sedimentability of the peptides (Table 1, ninth column)
generally agreed with what is expected from the topology predicted for
Ca
-ATPase. Note, however, that p14b and p43 (and
possibly also a small fraction of p29/30) were found both in the
soluble fraction and in the pellet, suggesting noncovalent interaction
of these peptides with the rest of the molecule.
The ATPase fragments formed
after proteolysis under the conditions of the Fig. 7A experiment were separated by gel electrophoresis. Both Laemmli
gels (not shown) and Tricine gels (Fig. 7B) indicated a
general stabilization of the long ATPase fragments (p81/83, p54, and
the intact ATPase itself) in the presence of 10 mM of either
Ca or Mg
. But Tricine gels showed
that 10 mM Ca
, and not Mg
,
exerted a specific stabilization of p28N and p27C fragments (see arrows) over long proteolysis periods (compare lane13 and lanes9 or 5 in Fig. 7B, see asterisks), with a concomitant
reduction in the amount of p19 formed.
Most peptides of low molecular
mass produced as a result of long term proteinase K treatment (running
at the front of Laemmli gels, as shown in Fig. 1, or separated
by SDS-PAGE in Tricine gels, data not shown) reflect further
degradation of the membranous peptides mentioned above, particularly in
the N-terminal region, which nevertheless, as in the case of SR
treatment with V8 protease (le Maire et al., 1993), left the
membrane-spanning segments M1-2, M3-4, and M5-6,
together with the attached stalk segments, intact. ()By
contrast, p19 was only slowly degraded, and, on a molar basis,
accumulated to a significant extent after 0.5-2 h of proteinase K
treatment. Thus, our proteinase K-treated SR membranes, in the latter
stages of proteolysis, are analogous to the 19-kDa membranes, which can
be obtained after proteolytic treatment of
Na
,K
-ATPase and
H
,K
-ATPase (Capasso et al.,
1992; Rabon et al., 1993). It is of note that accumulation of
ATPase fragments took place without proteolytic degradation of protein
components, known to be localized inside the SR vesicles (calsequestrin
and M55 glycoprotein; see Fig. 1and Fig. 7B).
These proteins were fully recovered in the membranous pellet obtained
after ultracentrifugation (see Fig. 2A), whereas they
were completely degraded as soon as the vesicular membrane was
disrupted, either by detergent solubilization or by alkaline EDTA
treatment (data not shown). Electron microscopy ultrastructural studies
(not shown) also indicated that the SR vesicular structure remained
intact after prolonged treatment with proteinase K. As a result, it can
be concluded that the cut between Met-817 and Asp-818 in SR vesicles,
leading to the p19 fragment, occurred on the exterior of the vesicles,
which corresponds to the cytosolic side. A similar result was obtained
with Na
,K
-ATPase (Karlish et
al., 1993), and both results, for P-type ATPases, are in agreement
with a cytosolic location of the M6-M7 loop (see also Mata et
al., 1992; Met al., 1993; Shin et
al., 1994), for which the exact location has been controversial in
Na
,K
-ATPase (Ovchinnikov, 1987;
Mohraz et al., 1994). Note, however, that the cleavage
observed between Leu-807 and Gly-808, leading to formation of p20, is
located unexpectedly close to the C-terminal border of the predicted
M6 segment, in fact just before it (e.g. Clarke et
al., 1990; Toyoshima et al., 1993).
In
addition, the intrinsic fluorescence experiments shown in Fig. 7suggest that the inability of Ca-ATPase,
proteolyzed under standard conditions, to retain Ca
binding properties reflects a lability of the bundle of
transmembrane fragments. This we conclude from the fact that when
proteolysis was performed in the presence of a high (10 mM)
Ca
concentration, Ca
binding to
ATPase fragments after 20-fold dilution of the proteolysis medium was
first retained, as shown by EGTA-induced fluorescence changes, but
chelation of Ca
irreversibly reduced the ability of
these fragments to rebind Ca
in a subsequent step
(compare single and doublearrowheads in columnc of Fig. 7A). This occurred
within a few seconds, implying that proteolysis after dilution had not
proceeded much further and thus was not responsible for the observed
loss in Ca
sensitivity. Since the presence of 0.3
mM Ca
during 80-min proteolysis, either in
the absence or the presence of Mg
(columnsa and b of Fig. 7A), was not
sufficient to retain the EGTA-induced fluorescence change, the
requirement for a high Ca
concentration presumably
derives from a need for keeping the Ca
binding cleft
permanently occupied by Ca
to avoid irreversible
inactivation after proteolysis. Thus, it appears that, in this respect,
proteolyzed SR membranes behave like
Na
,K
-ATPase 19-kDa membranes, for
which it has been clearly demonstrated that a major effect of
proteolysis is to increase the sensitivity to thermal inactivation, an
inactivation that is antagonized by cation occlusion within the
membrane-spanning segments, Na
being as efficient as
K
or Rb
in this respect (Or et
al., 1993; Shainskaya and Karlish, 1994). In fact, the
above-quoted work on trypsinized
Na
,K
-ATPase was a strong impetus for
us to perform the experiments illustrated in Fig. 6and Fig. 7. The rather similar relative effects of Ca
and Mg
in protecting
Ca
-ATPase during proteolysis at early times, between
0 and 12 min (Fig. 7), suggest that, at early stages of
proteolysis, Ca
and Mg
at high
concentrations are probably both recognized by a common site, e.g. the Mg
binding site in the catalytic center on
the cytoplasmic portion of the ATPase, whereas the significantly
different effects of these ions at later stages of proteolysis, between
30 and 80 min, suggest that only Ca
stabilizes the
ATPase polypeptide chain by binding to the Ca
binding
intramembranous cleft (see a related discussion with
Na
,K
-ATPase in Or et
al.(1993)). Note that Fig. 7B shows that in the
presence of 10 mM Ca
, 19-kDa peptides were
virtually absent throughout the 80-min proteolysis period, while the
membranous p28N and p27C peptides were stabilized, suggesting that
stabilization of the Ca
-ATPase polypeptide chain by
cations bound to the translocation sites seems to favor the formation
of Ca
-ATPase fragments slightly longer than those
resulting from ligand stabilization of the
Na
,K
-ATPase 19-kDa membranes.
In proteolyzed membranes,
thermal agitation probably allows larger movements of the transmembrane
segments with respect to each other than in the intact protein. Under
conditions where these transmembrane segments are not held together
properly oriented by cations bound to the translocation sites, they may
well change their relative orientations and fall into energy
``traps'' from which a return to the original cation binding
topography is either irreversibly lost or only occurs slowly ( Fig. 6and Fig. 7). Proteolyzed
Ca-ATPase fragments seem to be especially sensitive
to such thermal inactivation, while 19-kDa
Na
,K
-ATPase membranes with occluded
K
or Rb
evidently are more stable. It
is not easy to evaluate to what extent this is a qualitative or a
quantitative difference. Note in this connection that Ca
dissociates from intact Ca
-ATPase much more
rapidly (less than 100 ms half-time, see triangles in Fig. 4C) than K
or Rb
does from Na
,K
-ATPase. As the
chain in Na
,K
- or
H
,K
-ATPase is known to modulate
K
-dependent events, its presence may be important for
stabilization of the occluded form of these ATPases in both native and
proteolyzed membranes (see Jaisser et al., 1992; Schmalzing et al., 1992; Eakle et al., 1992; Capasso et
al., 1992; Lutsenko and Kaplan, 1993; Shainskaya and Karlish,
1994).
Irrespective of the detailed explanation for the difference
between Na,K
-ATPase and
Ca
-ATPase, it is clear from our results that cuts
outside the transmembrane region are deleterious for the subsequent
stability of the cation binding sites, implying that these sites are
very dependent on the conformation or rigidity of segments of the
protein outside the membrane. In fact, this is a necessary corollary of
the functional ion transport properties of intact ATPases, in which at
one point during turnover (corresponding to the release of occluded
ions to the other side of the membrane), the affinity of the binding
sites for cation is reduced and their topological orientation is
altered after phosphorylation of an aspartyl residue located far away
in the catalytic domain.