Drabikowski, Witold

Korczak, Bożena

Sarzała, M.Gabriela

Zubrzycka, Elżbieta

Elsevier - Publisher Connector 

Location of the peripheral proteins in sarcoplasmic reticulum vesicles 

Volume 90, number 2 FEBS LETTERS June 1978 
LOCATION OF THE PERIPHERAL PROTEINS IN SARCOPLASMIC RETICULUM VESICLES 
Elibieta ZUBRZYCKA, Boiena KORCZAK, M. Gabriela SARZAI;A and Witold DRABIKOWSKI 
Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Experimental. Biology, 
3 Pasteur Street, 02-093 Warsaw, Poland 
Received 4 April 1978 
1. Introduction 
The composition and the molecular organization 
of sarcoplasmic reticulum (SR) membrane from 
skeletal muscle has been investigated extensively for 
the last few years [I] . It is now well established that 
the protein pattern of this membrane is relatively 
simple. ln addition to the Cap, Mg2’-dependent 
ATPase (105 000 daltons), atypical integral membrane 
protein, SR from fast skeletal muscle contains also 
two peripheral proteins, called high affinity calcium 
binding protein (HACBP) (55 000 daltons) and 
calsequestrin (45 000 daltons) [l] . The function of 
two latter proteins is still not clear and their location 
in the membrane is a matter of controversy [2-71. 
The extraction of the SR vesicles with t e solution 
‘of EDTA leads to the release of both peripheral 
proteins [ 5 $1. EDTA, however, makes the vesicles 
leaky and permeable to macromolecules [8] ; there- 
fore, this approach cannot bring any information 
about the location of these proteins in the mem- 
brane. For that reason in the present work a non- 
penetrating chelating resin Chelex 100 was used 
instead of EDTA in order to localize the peripheral 
proteins in the SR vesicles. 
2. Materials and methods 
The light and heavy subfraction of SR vesicles were 
obtained from fast rabbit skeletal muscle as in [9]. 
Chelex 100 (13 1 0-Rad Laboratories, Richmond, 
CA) was prepared according to Seidel and Cergely 
[lo] . The vesicles of light or heavy subfraction 
suspended in 20 mM imidazole , pH 7.2 and 100 mM 
ElsevierjNorth-Holhnd Biomedical Press 
KCl (called throughout this paper imidazole-KCl 
buffer) at protein cont. 5 mg/ml were shaken for 
3 min with Chelex 100 suspended in the same buffer 
(10 mg Chelex 100/30 ml). Chelex 100 was removed 
by centrifugation at 1500 X g for 10 min and then 
the suspension was centrifuged at 100 000 X g for 1 h. 
In some experiments he vesicles of light or heavy 
subfraction suspended in the imidazole-KCl buffer 
were extracted with 1 mM EDTA at pH 8.0 for 
30 min at 0°C with constant stirring and then 
centrifuged for 1 h at 100 000 X g. The pellets and 
the supematants after Chelex 100 or EDTA treatment 
were used for the further studies. 
Sodium dodecyl sulphate-polyacrylamide g l 
electrophoresis (SDS-gel electrophoresis) n 7.5% 
slab-gel was carried out according to [ 1 l] at a current 
of 100 mA/plate. Protein content was determined 
according to [ 121 .
3. Results and diion 
SR vesicles can be separated [9] by an additional 
sucrose density gradient centrifugation i to 2 sub- 
fractions, the heavy one, consisting of normally 
oriented, right-side-out vesicles, and the light one 
containing most probably, inside-out vesicles. The 
protein pattern of both subfractions i virtually the 
same although they differ from each other in the 
relative proportions of calsequestrln a d HACBP 
(fig-l). 
ln the present work these 2 subfractions have been 
used for the studies on the location of the peripheral 
proteins. 
SDS-gel electrophoresis shows that the extraction 
215 
Volume 90, number 2 FEBS LETTERS June 1978 
123456 
Fig.1. Treatment of the vesicles of heavy (A) or light (B) SR subfraction with Chelex 100 or EDTA. Vesicles were treated with 
Chelex 100 or extracted with EDTA as in section 2 and then centrifuged for 1 h at 100 000 X g. The protein pattern of the 
supematants and the pellets was analyzed in SDS-slab gel electrophoresis carried out according to [ 111. (1) Intact SR vesicles; 
(2) proteins remaining in SR membrane after Chelex 100 treatment; (3) proteins released from the membrane by Chelex 100 
treatment; (4) calsequestrin purified according to [ 181; (5) pellet after EDTA extraction; (6) supernatant after EDTA extraction; 
105 K, Ca*, Mfl-ATPase; 55 K higher affinity calcium binding protein; 45 K, calsequestrin; 30 K, glycoprotein. 
with EDTA causes the release of both peripheral 
proteins from either subfraction (fig.1). In contrast, 
the treatment with Chelex 100 leads to the release of 
HACBP only from the heavy subfraction and of 
calsequestrin o ly from the light one (tig.1). 
The presence of calsequestrin i  the supematant 
obtained after Chelex 100 treatment of the vesicles 
of light subfraction and its absence in the supernatant 
obtained from the vesicles of heavy subfraction has 
been proved by the Ouchterlony double diffusion test 
[ 131 with the antiserum against calsequestrin (tig.2). 
It is known that the extraction of the SR vesicles 
Fig.2. Ouchterlony double diffusion test in an agarose 
plate. The well labeled AS contained antiserum from a sheep 
immunized against rabbit cakequestrin; the well labeled CS 
contained rabbit calsequestrin; the well labeled H and L 
contained the supematant after Chelex 100 treatment from 
the vesicles of heavy and light SR subfractions, respectively. 
216 
Volume 90. number 2 FEBS LETTERS June 1978 
with EDTA causes an increase of the activity of Ca*+, 
Mg*+-dependent ATPase [8] . Contrary to that Chelex 
100 treatment does not lead to any changes in the 
ATPase activity. This observation i dicates the lack 
of damage of the vesicles during Chelex 100 treat- 
ment. 
The results how that in normally oriented vesicles 
(present in the heavy subfraction) HACBP is located 
on the external surface of the membrane, whereas 
calsequestrin is bound to the inner surface. Moreover, 
the data furnish another evidence to the previous 
assumption [9] that the light SR subfraction contains 
the vesicles with the opposite orientation of the mem- 
brane than the vesicles present in the heavy sub- 
fraction. 
The results of this work indicating the internal 
location of calsequestrin i  normally oriented vesicles 
remain in agreement with the findings of many 
authors tudying the location of the peripheral 
proteins in SR [3-6,14,15] . Some authors [3-6] 
have published ata suggesting that HACBP is also 
present inside the vesicles. The results of this work 
show, however, that in right-side-out vesicles HACBP 
is bound to the outer surface of the membrane. 
The proteins present on the external surface of the 
vesicles of each subfraction, i.e., HACBP in the heavy 
subfraction and calsequestrin i  the light one, seem to 
be rather loosely bound. One can assume that the 
difference in the proportions of these 2 peripheral 
proteins between the subfractions ( ee fig.1) is due to 
the partial removal, during the purification procedure, 
of this protein, which is externally located in the 
given subfraction. This assumption may explain why 
the carefully washed preparations of SR obtained 
[15--l 71 contained virtually no HACBP, although 
this protein was present in the crude SR fraction. The 
most plausible xplanation is that HACBP, which 
according to the results of this work is located on the 
external surface of the vesicles, was removed uring 
the washing procedure. Scrupulous analysis of the 
densitometric tracings of the gels of the SR vesicles 
labeled with the nonpenetrating reagent diazonium 
salt of [35S]sulfanilic acid, presented [ 151, is also in 
favour of the external location of HACBP. Even in 
the case where there was virtually no visible protein 
band in the gel corresponding to HACBP, the incorpo- 
ration of the radioactivity in this region was quite 
high. 
Acknowledgements 
We wish to thank Professor David H. MacLennan 
from the University of Toronto for donating us the 
anti-calsequestrin serum. We also thank Mr Sfawomir 
Stachowski for his excellent echnical assistance. 
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217 


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Location of the peripheral proteins in sarcoplasmic reticulum vesicles

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