Tài liệu Báo cáo khoa học: The resident endoplasmic reticulum protein, BAP31, associates with

The resident endoplasmic reticulum protein, BAP31, associates

with c-actin and myosin B heavy chain

Analysis by capillary liquid chromatography microelectrospray tandem MS

Axel Ducret1

, Mai Nguyen2

, David G. Breckenridge2 and Gordon C. Shore2

1

Merck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Que´bec, Canada; 2

Department of Biochemistry,

McIntyre Medical Sciences Building, McGill University, Montreal, Que´bec, Canada

BAP31 is a 28-kDa integral membrane protein of the

endoplasmic reticulum whose cytosolic domain contains two

caspase recognition sites that are preferentially cleaved by

initiator caspases,such as caspase-8. Recently,we reported

that the caspase-resistant BAP31 inhibited Fas-mediated

apoptotic membrane fragmentation and the release of

cytochrome c from mitochondria in KB epithelial cells

(Nguyen M.,Breckenridge G.,Ducret A & Shore G. (2000)

Mol. Cell. Biol. 20,6731–6740). We describe here the char￾acterization by capillary liquid chromatography microelec￾trospray tandem MS of a BAP31 immunocomplex isolated

from a HepG2 cell lysate in the absence of a death signal.We

show that BAP31 specifically associates with nonmuscle

myosin heavy chain B and nonmuscle c-actin,two compo￾nents of the cytoskeleton actomyosin complex. Collectively,

these data confirm that BAP31,in addition to its potential

role as a chaperone,may play a fundamental role in the

structural organization of the cytoplasm. Here we also show

that Fas stimulation of apoptosis releases BAP31 associa￾tions with these motor proteins,a step that may contribute to

extranuclear events,such as membrane remodelling,during

the execution phase of apoptosis.

Keywords: apoptosis; BAP31; mass spectrometry; post￾translational modifications.

Apoptosis,or programmed cell death,is a physiological

mechanism by which multicellular organisms can eliminate

in an orderly fashion unwanted or damaged cells during

development,maturation or reparation [1]. Central to the

trigger of the apoptotic pathway is the activation of a family

of cysteine proteases,the caspases,which have been shown

to (in)activate a relatively large panel of proteins involved in

essential physiological functions. Cumulatively,these pro￾teolytic events disable homeostatic and repair processes,halt

cell cycle progression,mediate structural disassembly and

morphological changes,and mark the dying cell for

engulfment and elimination.

Recently,we identified a Bcl2/Bcl-XL and procaspase-8

associated protein,BAP31,a 28-kDa integral membrane

protein resident in the endoplasmic reticulum (ER) of most

if not all cell types [2–5]. Sequence analysis reveals that

BAP31 can be roughly divided in two domains (Fig. 1): a

hydrophobic 15-kDa N-terminal fragment is predicted to

form three transmembrane domains in which the short

hydrophilic N terminus and a 37 amino acid loop face the

lumen of the ER. The remaining 13-kDa domain,termin￾ated by the canonical KKXX ER localization signal,is

exposed to the cytosol [4]. Functionally,BAP31 has been

suggested to represent an ER-associated chaperone as it was

first detected associated with membrane-bound immuno￾globulin in lysates of B lymphocytes [6]. Consistent with this

proposed role,BAP31 can form transient associations with

newly synthesized IgD and cellubrevin as they exit from the

ER to the Golgi apparatus [5] and it has been recently

shown to participate in the quality control of the cystic

fibrosis transmembrane conductance regulator folding [7].

In addition,BAP31 has also been suggested to be

involved in apoptotis. It is capable of selectively recruiting

the procaspase-8 isoform,procaspase-8L,as well as a

predicted adapter protein,which promotes apoptosis. These

proteins in turn contribute to the ability of BAP31 to

associate with antiapoptotic Bcl2 family proteins,which

also make direct contact with the membrane-associated

N-terminal region of BAP31 ([4,8] and references therein).

In particular,Bcl2 has been demonstrated to block the cell

death pathway induced by expression of the E1A oncogene

[4,8]. In the absence of Bcl2, however, cell death signalling

leads to the activation of procaspase-8L and the resulting

proteolytic cleavage of BAP31 at two identical caspase-8

Correspondence to A. Ducret,F. Hoffmann-La Roche Ltd,

Roche Centre of Medical Genomics,Bau93/4.40,

Grenzacherstrasse 124,CH-4070 Basel,Switzerland.

Fax: + 41 61 688 1448,Tel.: + 41 61 688 9739,

E-mail: [email protected]

Abbreviations: ER,endoplasmic reticulum; LC-lESI-MS/MS,

liquid chromatography microelectrospray tandem

mass spectrometry; cr,caspase-resistant.

Proteins: BAP31 (CDM_human,accession number P51572); myosin

heavy chain nonmuscle type B (MYHA_human,accession number

P35580); myosin heavy chain skeletal muscle,fetal (MYH4_human,

accession number Q9Y623); nonmuscle actin c (ACTG_human,

accession number P02571).

Enzymes: trypsin porcine (TRYP_pig. accession number P00761;

EC 3.4.21.4).

(Received 19 September 2002,revised 19 November 2002,

accepted 26 November 2002)

Eur. J. Biochem. 270,342–349 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03395.x