STOMACH-SPECIFIC CALPAIN, nCL-2, LOCALIZES IN MUCUS
CELLS AND PROTEOLYZES THE β-SUBUNIT OF COATOMERCOMPLEX, β-COP
Shoji Hata1, Suguru Koyama1, 2, Hiroyuki Kawahara3, Naoko Doi1, 4, Tatsuya Maeda4, 5, Noriko
Toyama-Sorimachi6, Keiko Abe2, Koichi Suzuki7, Hiroyuki Sorimachi1, 4
1

Department of Enzymatic Regulation for Cell Functions, The Tokyo Metropolitan Institute ofMedical Science (Rinshoken), Tokyo 113-8613, 2Graduate School of Agricultural and Life Sciences,
University of Tokyo, Tokyo 113-8657, 3Graduate School of Pharmaceutical Sciences, Hokkaido
University, Hokkaido 060-0812, 4CREST, Japan Science and Technology (JST), Saitama 332-0012,
5

Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032,

6

Department of Gastroenterology, Research Institute, International Medical Center of Japan, Tokyo

162-8655, and 7New Frontiers Research Laboratories, Toray Industries Inc., Kanagawa 248-8555,
Japan
Running title: Possible function of stomach-specific calpain
Correspondence to Hiroyuki Sorimachi: Department of Enzymatic Regulation for Cell
Functions, Rinshoken, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan; E-mail:sorimach@rinshoken.or.jp; TEL/FAX: +81-3-3823-2181.
Calpain is a Ca2+-regulated cytosolicnCL-2-intracting molecules. Of these, theprotease. Mammals have 14 calpain genes,

β-subunit of coatomer complex (β-COP)

half of which are predominantly expressed

occurs in the stomach pit cells and isin specific organ(s); the rest are expressed

proteolyzed

ubiquitously. A defect in calpains causes

Furthermore,lethality/pathogenicity,

indicating

their

co-expressed in COS7 cells co-localized inphysiologicalindispensability.

the Golgi, and Ca2+-ionophore stimulation

identifiedacaused the proteolysis of β-COP near thewhoselinker region, resulting in the dissociation

physiological functions are unclear. To

of β-COP from the Golgi. These results

elucidate these, we characterized nCL-2 instrongly suggest novel functions for nCL-2

detail. Unexpectedly, nCL-2 was localizedthat involve the membrane trafficking ofstrictly to the surface mucus cells in the

mucus cells via interactions with coatgastric epithelium and the mucus-secreting

protein.

nCL-2/calpain-8a
stomach-specificwascalpain,

as

by

nCL-2

β-COP

in

vitro.

andnCL-2

goblet cells in the duodenum. Yeast
two-hybrid screening identified several

Calpain(EC

3.4.22.17)

is

an

1

intracellular Ca2+-regulated cysteine protease,skeletal-muscle-specificcomprising a superfamily in most organisms

CAPN3, are responsible for limb-girdle

including some bacteria, budding yeasts,

muscular dystrophy type 2A (9); and a single

fungi,

Calpains

nucleotide polymorphism in intron 3 of theproteolyze specific substrates at very limited

human gene for calpain 10, CAPN10, issites to irreversibly modify/modulate theirassociatedfunctions, structures, and activities, and are

Mutations in the calpain genes of various

thus called “modulator proteases”.

other organisms, including yeasts, plants,

plants,andanimals.

Fourteen

with

p94/calpain

type

2

diabetes(10).

calpain genes have been identified in humans

nematodes,

andand mice and can be classified into two

significant

phenotypes

categories according to their expression, i.e.,

defects in stress responses, sex determination,ubiquitousorganogenesis,ortissue-specific(1,2,3).

Drosophila,

3,

resultincorresponding

to

etc.

(11,12,13,14,15).

Ubiquitous µ- and m-calpains, two major

However, our understanding of the specificcalpains in mammals, form a heterodimer

molecular mechanisms underlying calpain

composed of a distinct 80 kDa catalytic large

functions in vivo is far from complete.

subunit (abbreviated to “µCL” and “mCL”,
respectively)

andacommonregulatory small subunit (30K).
three-dimensional

structures

One clue to the molecular actions ofkDa

calpain is their tissue-specificity, because thisCalpainshould reflect their specific cellular functions,30

define

four

which

are

highlydifferentiatedand(I–IV) and two (V and VI) domains in thespecialized in the corresponding tissues.

large and small subunits, respectively: theIndeed, a defect in skeletal-muscle-specificregulatory N-terminal domain (I), the proteasecalpaindomainmentioned above.

(II),

the

C2-domain-like2+

causesmusculardystrophy,

asnCL-2, also called

Ca /phospholipid-binding domain (III), thecalpain 8a, is another tissue-specific calpain,

penta-EF-hand domains (IV and VI), and thewhich is predominantly expressed in the

Gly-clustering domain (V) (4,5).

stomach (16).

In the2+

The domain structure ofabsence of Ca , the protease domain (II) isnCL-2 is identical to that of mCL describedfurther divided into two subdomains, IIa andabove.

IIb, which form a single active domain uponnCL-2, Capn8, generates nCL-2´ (also called

2+

By alternative splicing, the gene forcalpain 8b), which lacks domains III and IV

binding to Ca (6,7).
Genetic studies in mammals and other(16,17).

Possible functions have beenthereported for nCL-2 in the pituitary gland andphysiological indispensability of the calpains.

for its Xenopus laevis homologue, XCL-2, inA gene knock-out mouse with a mutation inembryogenesis

the gene for 30K, Capn4, exhibits embryonicspecific targets and functions of nCL-2 arelethality (8); mutations in the human gene forstill unclear.

organisms

haveclearlyshown(18,19).

However,the2Another tissue-specific calpain, nCL-4,

is essential first to determine its localization

the expression of which is predominantly inin the stomach.

the digestive tract, also occurs in the stomach.

hybridization

It is reported to be down-regulated in a subset

immunohistochemical analysis of nCL-2/-2´of gastric cancer patients and is involved inwere performed to localize them precisely.

the suppression of tumorigenesis (20,21,22).

Surprisingly, nCL-2 was strictly localized inµ-Calpain is also involved in the acidthe pit cells, in sharp contrast to thesecretory process by cleaving ezrin, aexpression

membrane cytoskeletal protein, in parietal

Furthermore, yeast two-hybrid screening

cells (23).

followed

How these data relate to the

In this study, in situ
(ISH)

of

the

by

and

ubiquitous

calpains.

biochemical

analysisproteolyticinteraction

physiological functions of nCL-2/-2´ has yet

demonstrated

to be elucidated.

between nCL-2 and the β-subunit of the COPIThe stomach is a highly differentiated organ

coatomerwithcomplexsuggesting novel functions for nCL-2 that

programmed glandular structures, organizedinvolve the membrane trafficking of mucus

into diverse types of epithelial cells.

cells by interacting with coat proteins.

specific

functions

and

The

the

complex(β-COP),stronglyrodent stomach is composed of four regions:fundus, cardia, corpus, and pylorus, the latter

MATERIALS AND METHODS

three of which have numerous tubular pits
with glands deep within them (24).

The pits

Materials – Adult male Wistar rats andare lined by pit cells that secrete neutral

C57BL6/J mice were sacrificed by cervicalmucus to protect the epithelium from aciddislocation to obtain tissue samples for ISH(25).

The oxyntic glands in the corpus

and immunohistochemistry. Tissue samples

consistofacid-secreting

pepsinogen-secreting
hormone-secreting(25,26).

parietalcells,

were removed, washed in ice-cold PBS,

cells,

andsliced,enteroendocrine

cells

compound (Sakura Finetechnical Inc., Tokyo,

chief

Pylorocytes and gastrin-secreting G

Japan),

embeddedandinfrozenTissue-TekinliquidO.C.T.
nitrogen.

cells are mainly found in the pyloric glands ofSections (7 µm thick) cut by cryostat were

the pylorus (27).

placedAll these cells originate

on

glass

slidesfrom proliferative progenitor cells located3-aminopropyltriethoxysilane

under the pits and undergo continuous

Glass

renewal.

immunocytochemistry,

The progenitor cells follow a

Inc.,

Osaka,

coated(Matsunami

Japan).
COS7

with

cells

For
were

specific differentiation process during theirplated onto Lab-tek chamber slides (Nalge

migration upwards, downwards, or both, to

Nunc International Inc.).

their final programmed destinations (25).

analysis, the gastric mucosa was scraped from

Therefore, to understand nCL-2 functions, it

the stomach and homogenized in buffer A (20

For western blot3

mM Tris-Cl ,pH 8.0, 5 mM EDTA, and 1 mM

antibodies

wereraisedinrabbitsandDTT) containing 0.2 mM PMSF, 10 µg/ml

affinity-purified

using

recombinantrat

aprotinin, and 50 µM pepstatin A.

The

nCL-2´ (full-length) and an nCL-2-specificcentrifugation

region (amino acids 379–713), respectively.

supernatantrecoveredby(20,000 × g, 30 min, 4°C) was used as theprotein sample.

µ-Calpain was prepared as

Anti-H+/K+-ATPaseβ-subunit

monoclonal

antibody

2G11),

anti-PCNA(clonedescribed previously (28).

monoclonal

ISH – cDNA fragments encoding mouseanti-GM130 monoclonal antibody (clone 35),nCL-2 (2,020 bp, nt 250–2269), mouse nCL-4

anti-β-COP

antibody

and(1,369 bp, nt 700–2068), mouse mCL (1,491

monoclonal

antibody(clonebp, nt 700–2190), and mouse µCL (1,555 bp,

purchased from Sigma, Neomarkers, BD

nt 700–2254) were amplified by PCR using a

Biosciences,

mouse

Stratagene

17-dayembryocDNAlibrary

antibody

AffinityInc.,(clonePC-10),
anti-FLAG

M2)

were

Bioreagents

respectively.

and
Alexa

(CLONTECH Inc., ML4013AH) or mouse488-conjugated goat anti-mouse and Alexa

stomach first-strand cDNA and the following

594-conjugated goat anti-rabbit IgGs weresetsfrom Invitrogen Inc.

ofprimers:(nCL2-s)

5´-ctggacaaactcaaggcatcatctggaa-3´

andImmunostaining – Stomach sections and(nCL2-as) 5´-agggaagttgttgatgtccagagggtc-3´;

COS7 cells prepared as described above were

(nCL4-s)fixed with 4% paraformaldehyde in PBS.

5´-gctctagataccctgaatgcctcagaatctgaa-3´andAfter incubation with 5% normal goat serum,(nCL4-as)

1% BSA, and 0.1% Triton X-100 in PBS,

5´-gcggatcctcagatgttcatggtcaggctgatg-3´;they were incubated with primary antibody at(mCL-s)4°C overnight, and then with secondary

5´-gctctagaaggctttggagaaaggttctctgct-3´andantibody at room temperature for 30 min.

(mCL-as)An

5´-gcggatcctcaaggtccagatacaaagtgtcta-3´;Vectastain Elite ABC kit (Vector Laboratories(µCL-s) 5´-gctctagacctgcagaaggccccc-3´ andInc.) and 3,3´-diazobenzidine hydrochloride(µCL-as)or an immunofluorescence method using

5´-gcggatcctcaggcaaacatagtcagctggagc-3´.

Alexa-labeled secondary antibodies was used

The amplified fragments were subcloned into

to detect the bound antibodies.

the pBluescript SK(–) vector.

Digoxigenin

Northern blot analyses – Mouse RNAs were(Dig)-labeled riboprobes were synthesized

isolated and blotted onto nylon membranes as

anddescribed previously (16).

hybridized

accordingtothe

HRP-catalyzing

RNA-blotted

methodusing

theFor human

manufacturer’s manual for nonradioactive intissues,membranewassitu hybridization (Roche Inc.).

purchased from CLONTECH Inc. (nos.

Antibodies – Anti-nCL2/2´ and -nCL2C

636818 and 636835).

Probes were prepared4

from cDNA clones for mouse and humanwere inserted into the pCold I vector (TaKaRa

nCL-2,

Inc., Kyoto, Japan) to produce proteins withmouse

glyceraldehyde

nCL-2´,

mCL,

phosphateanddehydrogenase

an N-terminal His-tag.

For COS7 cell

(GAPDH), and bands were detected astransfections, cDNAs were inserted into

previously described (16).

pSRD (29) to produce proteins with an

Yeast two-hybrid analyses – Full-length

N-terminal FLAG or HA tag.

mouse nCL-2 or domain IV (corresponding toProtein expression and purification – The E.

amino acids 533–713) was cloned intocoli expression plasmids constructed as

pGBKT7 (CLONTECH Inc.) and used as bait.

described above were transformed into E. coli

The bait plasmid and then a mouse 17-day

BL21/pG-Tf2

embryo cDNA library (ML4013AH) weretransformants were cultured at 37°C in 500

transformed into Saccharomyces cerevisiae

ml of LB medium containing 0.1 mg/ml

AH109, according to the manufacturer’s

ampicillin and 5 ng/ml tetracycline to A600 =instructions.

0.5.

Cells

were

platedonto(TaKaRa

Inc.),

andtheProtein expression was induced by the

SD-LWHA plates and incubated at 30°C.

addition of 0.5 mM IPTG for 24 h at 15°C.

Prey plasmids were recovered from the grown

Harvested cells were suspended in 40 ml ofcolonies by transformation of the isolated

buffer B (20 mM Tris-Cl , pH 7.5 and 150

total yeast DNA into E. coli, and themM NaCl), and lysed with a French press

sequences were determined.

(American

Instrument

Inc.).

The

Cloning and construction of expression

supernatantwasrecoveredbyplasmids – Mouse stomach total RNA was

ultracentrifugation (105,000 × g, 30 min, 4°C),

extracted

and filtered through 0.22 µm pore filter

from

an

adult

male

mouse

(C57BL6/J) using Trizol reagent (Invitrogen(Millipore Inc.).

Inc.).

were

applied to a 2 ml Ni2+-chelating agarose

reverse-transcribed from 5 µg of total RNA

column (Qiagen Inc.) equilibrated with bufferusing a First-Strand cDNA Synthesis Kit

B.

(Amershamimidazole in buffer B and eluted with 200

First-strand

Biosciences

cDNAsInc.).

Mouse

The supernatant was thenThe column was washed with 20 mM

nCL-2, β-COP, Eya2, GPS1, nm23-M2, andmM imidazole in buffer B.

nm23-M4 cDNAs were amplified by PCR

further purified by MiniQ anion-exchangeusing Pfu DNA polymerase (Stratagene Inc.)

columnand the synthesized first-strand cDNAs

chromatography with a linear gradient of

The β-COP deletion

0–0.5 M NaCl in 10 mM Tris-Cl (pH 7.5), 1

described above.

(Amersham

The eluent was

Biosciencesmutant, β-COP-C (amino acids 529–953)

mM EDTA, and 1 mM DTT.

cDNAs, was prepared by PCR using Pfu

was

DNA polymerase and the full-length β-COP

homogeneity.

cDNA.

stored at 4°C until use.

For bacterial expression, cDNAs

confirmed

by

Inc.)

Purification

SDS-PAGE

forits

The purified protein was

5Proteolysis assay – The pSRD constructs

supernatant of COS7 cells transfected with

were

FLAG-tagged

transfected

into

COS7

cells

by

mouse

nCL-2:C105S

waselectroporation, as previously described (29).

prepared by homogenizing cells with lysis

For the in vitro assay, COS7 cells werebuffer and centrifugation at 20,000 × g for 15

harvested 48 h after transfection, suspendedmin.

in buffer A, and lysed by sonication.

µg of GST or GST–TOM70 immobilized onThelysates were then incubated with a final
concentration of 5 mM Ca
min at 30°C.

2+or EDTA for 30

The reactions were stopped by

the addition of 6 × SDS sample buffer.

For

the cell culture assay, cells were treated with
2+

The supernatant was incubated with 5

glutathione–Sepharose

4B(AmershamBiosciences Inc.) for 3 h at 4°C.

The beads

were washed five times with lysis buffer, andmixed with SDS sample buffer.
Protein sequencing – After SDS–PAGE, themedium containing 10 µM Ca

ionophore,

proteins

A23187, 48 h after transfection.

Cells weremembranewereblotted(Appliedonto

Pro-Blot

Biosystems

Inc.).

harvested 20 h after A23187 treatment (at thisTarget protein bands on the membrane werepoint, cells were not significantly damaged

visualized by Coomassie Brilliant Blue G-250

under our culture conditions), suspended instaining, excised, and washed three times

buffer A, and lysed by sonication.

The

with 0.1% (v/v) trifluoroacetic acid in 50%

lysates were mixed with 6 × SDS sample(v/v) methanol and then with absolutebuffer and boiled.

methanol.

The samples were used

Sequencing was performed withfor SDS–PAGE and western blot analysis.

the ABI 491cLC protein sequencer (Applied

Immunoprecipitation and GST pull-down –Biosystems

For immunoprecipitation, transfected COS7

manufacturer’s instructions.

Inc.)accordingtothecells were harvested 48 h after transfection
and lysed with lysis buffer (buffer A

RESULTS

containing 5 mM EDTA, 0.5% nonidet P-40
(NP-40), 0.2 mM PMSF, and 10 µg/ml

nCL-2 is expressed specifically in the pit cells

aprotinin).

The supernatants were recoveredof the gastric mucosa – To investigate theby centrifugation at 20,000 × g for 15 min

localization of nCL-2/-2´ expression in theand incubated with a 50% slurry of FLAG

stomach, in situ hybridization (ISH) analysis(M2)of the corpus and pylorus of the mouse

agarose(Sigma

Inc.)

for

2

h.

Immunoprecipitated proteins were eluted withstomach was performed.

0.1 mg/ml FLAG peptide and mixed withexpressed around the upper quarter of the

SDS

corpus (oxyntic) mucosa and the upper half ofsamplebuffer.

TOM70

cDNA

identified by yeast two-hybrid screening wasthesubcloned into pGEX-5X to produce a

mucus-secreting pit cells are abundant (Figs.

GST-fusion

1A, B, 2A, B), whereas µCL and mCL

protein.

The

cell

lysatepyloric

mucosa,nCL-2/-2´ was

where

the

6

transcripts were detected in all cells including

for progenitor cells.

proliferative cells of the pyloric mucosa (Figsoverlap with that of nCL-2, indicating that

1D, E, 2D, E).

nCL-2 is localized specifically in the pit cells

The digestive-tract-specificcalpain, nCL-4, showed expression similar to

These signals did not

and is thus implicated in pit cell functions.

but a little wider than that of nCL-2 (Figs. 1C,
2C).

nCL-2 is expressed in a subset of goblet cells
The stomach was analyzed using

in the small intestine – Northern blot analysis

anti-nCL2/2´ (reactive to both nCL-2 and -2´)of human and mouse tissues with long

and anti-nCL2C (reactive only to nCL-2)

exposures revealed that the gene for nCL-2 isantibodies (Fig. 3A).

expressed at low levels in the trachea andFirstly, western blot

analysis using protein extract from the gastricintestines (Fig. 4A).

mucosawere treated with ISH to detect the expression

showedthat

theanti-nCL2/2´

Therefore, these tissues

antibody recognized nCL-2 but not nCL-2´

of nCL-2 and nCL-4 mRNAs.

(Fig. 3B), indicating undetectable or noobserved in some epithelial cells scattered in

expression of nCL-2´ protein in the gastric

the villi of the duodenum (Figs. 4B, C).

mucosa, even though the amounts of nCL-2

Furthermore, the nCL-2-positive cells wereand -2´ mRNAs in the stomach were similar

scattered in the upper half of the villi close to(seeConsistently,

the luminal surface (Fig. 4B), whereas mostimmunohistochemical analysis showed thatof the nCL-4-positive cells were in the lower

anti-nCL2/2´ andregions close to the crypt (Fig. 4C).

Fig.

4A)

(16).

anti-nCL2C

antibodies

Signals wereidentified identical signals (Figs. 3C, D),

Immunohistochemical

analysisusingsuggesting that signals for nCL-2´ were alsoanti-nCL2/2´

showedpatterns

undetectable in situ.

consistent with those obtained with ISH (Fig.

The localization of nCL-2 protein was

4D).

antibody

The nCL2/2´-positive cells had anconsistent with that observed by ISH (Figs.

expanded shape in the apical region and were

3C–F).

stained in the basal region.

Significantly, nCL-2 signals were

Their scatteredpositive in the relatively smaller cells in the

distribution and the shape of the cells in thepits, and were absent from larger cells (Fig.

intestinal

3F, arrowheads), which are considered to becorrespond to mucus-secreting goblet cells.

the parietal cells located in the pit region (25).

ISH and immunohistochemical analysis of the

To confirm this, stomach sections weretrachea, esophagus, and small and large

doubly immunostained with antibody directed

intestines produced no significant signals++

epitheliumindicate

that

they

against H /K -ATPase (green cells in Fig. 3G),

(unpublisheda marker protein for parietal cells, or one

expression levels were below the sensitivity

directed against proliferating-cell nuclearof our detection method.

data),

probably

becauseantigen (PCNA) (green in Fig. 3H), a marker

7

nCL-2 interacts with GPS1, β-COP, nm23-M2,

expression system and pull-down assays (Figs.

nm23-M4, and Eya2 – To elucidate the

5E, F).

physiological functions of nCL-2 in pit cells

proteins interacted with the penta-EF-handatpossible(PEF) domain IV of nCL-2, which isnCL-2-interacting proteins were investigated.

consistent with previous reports that thisthemolecular
5level,
6

It should be noted that theseWith 4.2 × 10 and 10 clones used as baitsdomain functions in the recognition ofcorresponding to domain IV and full-length

substrates

nCL-2,(32,33,34,35).

respectively,

yeast

two-hybrid

andregulatory

molecules

screening of a mouse 17-day embryo cDNA

Eya2 is one of the highly conserved

library identified 19 and 142 prey clones,

vertebrate homologues of the Drosophila eya

respectively (table 1).

Neither domain IV(eyes absent) gene product, required for thenor full-length nCL-2 identified 30K, whereas

proper development of compound eyes.

our previous screening of other cDNA

Eya2 consists of an N-terminal transactivation

libraries with baits corresponding to domain

domain and a C-terminal Eya-conserved

IV and full-length µCL isolated several 30K

phosphatase domain (Fig. 5A).

clones (30).

A two-hybrid assay using

two-hybrid assays using truncated cDNA

full-length nCL-2 and 30K showed noclones indicated that the binding site forbinding under the same conditions used whennCL-2 is in the Eya domain (Fig. 5A).

µCL and mCL bound 30K (unpublished data),

GPS1 (also called SGN1/CSN1/COPS1) isstrongly suggesting that nCL-2 exists as aone of the subunits of the COP9 signalosome,monomer/homo-oligomer without 30K, as isa multifunctional protein complex conserved

the case for p94 (31).

among

Among

the

molecules

identified,eukaryotes

tricopeptide

repeat

(36).
(TPR)

Yeast

GPS1

has

and

PCI

vimentin and desmin were not focused(proteasome, COP9 and eukaryotic translation

regardless of their abundance in the obtained

initiation factor 3) domains (37,38) in the N-

preys, because vimentin is not expressed inand C-terminal halves, respectively.

differentiated epithelial cells such as the pit

latter is the binding site for nCL-2 according

cells and desmin is mainly expressed into the structure identified by screening (Fig.

muscle cells.

5B).

Among the rests, Eyes absent

The

The NDPKs, nm23-M2 and nm23-M4,

homologue-2 (Eya2), G-protein pathway

are members of the nm23 family of

suppressor-1 (GPS1), nucleoside diphosphateoligomericDNA-binding

kinases

nucleoside

diphosphate(NDPKs;nm23-M2

and

-M4),

regulate

diverseproteins

withkinaseactivity.

cellulareventstranslocase of outer membrane 70 (TOM70),

They

and the β-subunit of the COPI coatomer

including growth and differentiation, and arecomplex (β-COP) were confirmed in theirimplicated in the pathogenesis and metastasis

interactions with nCL-2 using a COS7 cellof tumors (39).

TOM70 is a mitochondrial

8

protein, one of the components of the

β-COP is expressed in the proliferative andtranslocase of the outer membrane (TOM),

pit cells, and shows proteolytic interaction

and functions to import proteins destined forwith nCL-2 – Because the expression andall submitochondrial compartments (40).

localization of the proteins identified above in

TOM70 has TPR domains and several of

the stomach have not yet been reported, ISH

them are binding sites for nCL-2 (Fig. 5C).

analyses were performed.

The predominant

β-COP is a subunit of the coat protein I

expression of β-COP and Eya2 was in the(COPI) coatomer protein complex, one

upper one-third of the oxyntic mucosa and in

representative of the intracellular vesicle

most regions of the pyloric mucosa (Fig. 6).

coats that include COPII and clathrin.

The

In contrast, the expression of GPS1, TOM70,

COPI complex contains seven subunits (α-,and NDPKs was observed in the base regionβ-, β´-, γ-, δ-, ε-, and ζ-COPs) and a smallofGTPase, ADP-ribosylation factor 1 (ARF1).

(unpublished data), suggesting that their

COPI-coated vesicles are involved in protein

localization is distinct from that of nCL-2 intransport within the Golgi apparatus and invivo.

retrograde selective transport from the Golgi

theoxynticandpyloric

mucosae

To investigate whether or not the

From the N-terminus,

observed interactions indicate that they are

β-COP is composed of an adaptin N-terminal

substrates of nCL-2, we co-expressed GPS1,

domain, a linker region, and a potential “ear”

NDPKs, β-COP, or Eya2 with wild-type or a(also called “appendage”) domain (Fig. 5D)

protease-inactive(42,43).

The ear domain is found in theactive-site missense mutation) in COS7 cells.

C-terminal region of γ-COP, another COPIThe cell lysates were then incubated with

γ-COP

Ca2+ or EDTA to test for Ca2+-dependent

to the ER (41).

subunit.

shows

high

mutantnCL-2(C105S,

three-dimensional similarity to the C-terminal

proteolysis by nCL-2.

regions of α- and β-adaptins (also called α-neither GPS1 (lanes 1–4), nm23-M2 (lanes

and β-AP2), which interact with several

5–8), nm23-M4 (lanes 9–12), nor Eya2 (lanes

proteins such as clathrin.

Although the

13–16) produced any proteolytic fragment,

sequence similarity between the C-terminal

even with wild-type nCL-2 in the presence ofregions of the β- and γ-COPs is low, they

Ca2+ (lanes 1, 5, 9, and 13).

share a highly conserved FxxxW motif,

was co-expressed with wild-type nCL-2 (Fig.

suggesting the presence of a putative ear

7B, lanes 1 and 2), proteolytic fragments

domain in β-COP (42).

The structures of thewere produced in a Ca2+-dependent manner.

isolated cDNA clones indicate that thisProteolysis was suppressed by the addition of

putative ear domain is the binding site forinhibitors of calpain: E64c (lane 3), calpain

nCL-2 (Fig. 5D).

inhibitor I (lane 4), calpeptin (lane 5), andMG132 (lane 6).

As shown in Fig. 7A,

When β-COP

Furthermore, when β-COP

9was co-expressed with protease inactivenCL-2 (lanes 7 and 8), proteolysis did not
These results indicate that β-COP is

occur.

β-COP and nCL-2 are co-localized in the
Golgi

–

As

described

above,

β-COP

proteolyzed directly or indirectly by nCL-2

associates with COPI-coated vesicles related

protease activity in vitro.

to the Golgi membrane, and is thus localized

To

test

whethernCL-2

directlyin the Golgi apparatus (44).

When nCL-2

proteolyzes β-COP, we expressed β-COP andand β-COP were co-expressed in COS7 cells,

nCL-2 in Escherichia coli and purified themboth proteins were detected densely in theusing His-tag affinity and anion-exchange

Golgi stacks, and their expression overlapped

columnextensively

chromatographies.

When

(Fig.

8A,

a–f).

Similarrecombinant β-COP was incubated withlocalization of nCL-2 was observed in COS7

nCL-2, two major proteolytic fragments (62cells transfected with nCL-2 alone (Fig. 8A,

kDa and 47 kDa) of β-COP were produced in

g–l).

2+

To confirm the localization of nCL-2

a Ca -dependent manner (Fig. 7C, lanes 1–9,

in the Golgi, antibody directed against

closed arrowheads), which were not detected

GM130, a cis Golgi marker, was used forin the sample without β-COP (lane 10).

doubleThe

staining.

Consistently,

themolecular mass and the presence of theexpression of nCL-2 and β-COP was detected,

His-tag strongly suggests that the 62 kDa

overlapping the localization of GM130

band corresponds to the N-terminal fragment

expression (Fig. 8A, g–o).

observed in the COS7 cell experiments (Fig.

indicate that nCL-2 and β-COP interact in the

7B, lane 1, open arrowhead), and that the 47

Golgi stacks.

These results

kDa band corresponds to the remaining
The

Identification of β-COP as an in vivo

N-terminal sequence of the 47 kDa band wassubstrate of nCL-2 – To investigate thedetermined, and β-COP was shown to beinteractions between β-COP and nCL-2 underproteolyzed near the C-terminus of theconditions similar to in vivo conditions, COS7

adaptin N-terminal domain, between Ser528

cells transfected with nCL-2 were treated with

C-terminal

fragmentand Ser529.

of

β-COP.

Quantification of the β-COP

the Ca2+ ionophore, A23187.

As shown inbands clearly showed very rapid proteolysis

Fig. 8B, one major proteolytic fragment (60by nCL-2 (Fig. 7C, graph).

kDa) of endogenous β-COP was detected

These results indicate that β-COP is2+

only when COS cells were transfected withproteolyzed by nCL-2 in a Ca -dependentwild-type nCL-2 (lane 4, closed arrowhead).

manner.

The

This is the first report of theproteolysis of

β-COP.

molecular

mass

of

the

fragment

Therefore, thecoincided with that detected by in vitro

interactions between these molecules were

proteolysis (Fig. 8C, closed arrowhead).

further investigated.

The 60 kDa fragment was not observed when

10

transfectedwithprotease-inactive

and of the proteolysis of the COPI subunit.

nCL-2:C105S (Fig 8B, lane 6), indicating that

The localization of nCL-2 was strictly

β-COP is proteolyzed by nCL-2 in living

restricted to the pit cells in the stomach and to2+

cultured cells after stimulation with Ca

inthe goblet cells in the upper half of the villi ofthe duodenum.

the same manner as observed in vitro.

The pit and goblet cells

originate from progenitor cells at the isthmus
The proteolyzed fragments of β-COP were notand the base region of crypts, respectively,

localized to the Golgi – To investigate thethen migrate upward to the lumen.

physiological significance of the limited

cells are dedicated to secreting mucus, which

proteolysis of β-COP described above, the N-

is one of the possible functions involving

and C-terminal proteolytic fragments of

nCL-2.

In the duodenum, the distributions

β-COP (β-COP-N and -C, respectively) were

of

nCL-2

expressed

cells.

digestive-tract-specific calpains, are clearly

Immunocytochemical analysis revealed that

distinct, in that nCL-4 is expressed in the

β-COP-N and -C showed rather diffuse

goblet cells in the lower half of the villi.

expression patterns that partially overlapped

Although goblet cells are reported to containthat of GM130 in the Golgi (Figs. 9A–I).

several different types of cells (45,46), the

β-COP-N and -C expressed in COS cells did

distributions

not affect cell growth and viability(data notcell-stage, rather than cell-type, specificity,

These results strongly suggest thati.e., nCL-2 functions at late stages, when cells

the limited proteolysis of β-COP by nCL-2

are terminally differentiated, whereas nCL-4

triggers translocation of the β-COP fragmentsfunctions at early to mid stages, in processes

from the Golgi to the cytosolic space.

including the proliferation and differentiation

shown).

inCOS7

of cells.
DISCUSSION

and

of

nCL-4,

thesecalpains

These

the

suggestIn the stomach, the localizations of

nCL-2 and nCL-4 do not differ, except thatthe distribution of nCL-4 is slightly broader

In this study, we have demonstrated

than that of nCL-2.

This is consistent withthat the “stomach-specific” calpain nCL-2 isthe idea discussed above that nCL-4 functions

expressed specifically in the pit cells of thein those cells close to the stem cells.

stomach and also in subsets of goblet cells in

However, the precise roles shared by thesethe duodenum, and that nCL-2 proteolyzes

calpains must be examined further.

β-COP, a subunit of the COPI coatomer
complex, in the Golgi upon stimulation with
2+

In

contrasttotherestricted

localizations of nCL-2 and nCL-4 in the

Ca , resulting in the dissociation of β-COP

oxyntic mucosa, conventional ubiquitous µ-from the Golgi.

This is the first report ofand m-calpains show rather diffuse and very

calpain localization in gastrointestinal tissues

little expression in this region (see Figs. 1D,

11

E).

In

the

pyloric

mucosa,these

and/or scaffold protein for nCL-2.

Eya2 wasexpressed

first identified as one of the highly conserved

ubiquitously, and especially abundantly in thevertebrate homologues of the Drosophila eyes

pit and the isthmus (see Figs. 2D, E).

absent gene product, required for the proper

conventional

calpains

are

These

sharp contrasts strongly suggest that thedevelopment of compound eyes.

functions of the tissue-specific calpains in

expressed in the cytoplasm of various tissues

gastrointestinalberelatively late in mammalian development.

compensated by the conventional calpains, at

Following its translocation to the nucleus,

least in the oxyntic mucosa.

Eya2 forms a synergistic complex with thetissuescannot

It is

Yeast two-hybrid screening followed

DNA-binding homeodomain-containing Sineby in vitro pull-down analysis identified

oculis 1 (Six1) protein and a transcription

several nCL-2-interacting proteins, including

factor, Dachshund 2 (Dach2).

TOM70, GPS1, nm23-M2, nm23-M4, Eya2,

a transcriptional co-activator and its tyrosine

and β-COP.

phosphatase activity regulates specific target

ISH analysis showed that, of

It thus acts asthese, only Eya2 and β-COP are expressed ingenes

the pit cells of the oxyntic and pyloric

differentiation,

death

duringmucosae, suggesting an in vivo interaction

mammalian organogenesis (48,49).

This

with

physiologicalstudy shows, for the first time, the limited

significance of the observed interactions

expression of Eya2 in the progenitor and pit

betweencells of the stomach.

nCL-2.
nCL-2

Thus,
and

the

TOM70,

GPS1,

controlling
and

cell
cell

proliferation,

It is possible that

nm23-M2, and nm23-M4 is not clear at

nCL-2, binding to Eya2, is involved in genepresent.

regulation during pit cell maturation in the

However, it should be noted thatconventional

calpains

skeletal-muscle-specificcalpain,andstomach.

p94,

The

proteolyticinteraction

andproteolyze HSP60, a major chaperon protein

co-localization of nCL-2 and β-COP in thein the mitochondria (47).

Proteolysis of

Golgi strongly suggests novel functions for

HSP60 was observed not only in vitro, but

nCL-2 in intracellular membrane trafficking.

also in living cultured cells, suggesting a

β-COP is a subunit of the COPI coatomer

physiological significance (29).

Therefore,

complex, which coats the Golgi-derived

the interaction between nCL-2 and TOM70

vesicles involved in protein transport from theimplies a novel but as yet unidentified

Golgi to the ER and within the Golgi.

function of the calpains in mitochondria.

a COPI-coated vesicle reaches the acceptor

When

An in vitro assay using a COS7 cell

membrane, GTP hydrolysis by the small

expression system showed that β-COP, but

GTPase, ARF1, triggers coat disassembly,

not Eya2, is a proteolytic substrate for nCL-2.

allowing vesicle fusion (50).

This suggests that Eya2 is instead a regulatorycomplex can be reversibly dissociated into

The COPI

12

two subcomplexes, F-COPI composed of β-,dissociate from the Golgi, and resulting in theγ-, δ-, and ζ-COPs, and B-COPI composed ofirreversible disassembly of the coat proteins

α-, β´-, and ε-COPs (51).

Sequence(Fig. 10).

similarities, as well as biochemical andThis

nCL-2-mediated

COPI

structural biological properties, indicate thatdisassembly model is consistent with thethereport that calpain may be involved in the

F-

and

B-COPI

complexesaresignificantly similar to the adaptin andmaturation

clathrin complexes, respectively, other major

clathrin–adaptin uncoating by the limited

vesicle-coating proteins involved in transport

proteolysis of clathrin and the adaptins (54).

between the trans Golgi and the plasma

Moreover,membrane (42).

β-COP, like γ-COP and theofsecretorythegranulesrelationshipvia

betweenmembrane trafficking and the calpains has

adaptins, can be divided into three regions: anbeen

N-terminal α-helical domain called the

localization in the ER and Golgi (55), in lipid“adaptin N-terminal domain”, a flexible linker

rafts (56), and in multivesicular bodies (57)region, and a C-terminal ear domain (see Fig.

etc., suggesting a general involvement of

5D).

various calpains in a variety of membraneItshould

be

noted

that

the

confirmed

trafficking

recently,

processes.

e.g.,

calpainContinued

nCL-2-binding site of β-COP is in the earanterograde protein transport in the secretory

domain, and that the proteolytic site of β-COP

pathway(Ser528–Ser529) is located near the boundary

COPI-mediated retrograde, retrieval transport

between the N-terminal and the linker

system. The stomach is an “extreme”

domains (see Fig. 5D).

The linker domains

environment where the pH is extremely lowof β- and γ-COP bind to the WD40 repeat

and many infective microorganisms enter, anddomains in β´-COP and α-COP, facilitating

extensive protection by a tough mucosa isthe assembly of the F- and B-COPI

thus essential.

subcomplexes, and regulatory or accessory

amount of glycoproteins secreted from theproteins such as AP180, eps15, amphiphysin,

mucosa, such as mucins, a secretion systemand epsin interact with the ear domains of thethat includes retrograde transport has toadaptins and probably with that of β-COP

function at full capacity.

(42,52,53). Although the mechanisms of thereason why the stomach has an additional

disassembly of the COPI and clathrin–adaptin

COPI disassembly system involving nCL-2.

complexes are not fully understood, theThe different levels of expression of nCL-2 in

β-COP

the intestines and trachea, in which theprovides a new model for COPI disassembly:

mucosa is also very important, possiblynCL-2, binding to the ear domain of β-COP,

explain the requirement for this extra

cleaves the linker region, causing β-COP tosecretion system in the stomach, mediated byinteraction

between

nCL-2

and

is

tightlycoupledwith

To ensure the quality andThis may be one

13

nCL-2, in addition to the usual system.
It is also possible that, like Eya2,

kinase cascades, is a strong possibility,
although there are few reports of scaffold

β-COP functions as a scaffold protein forproteins

nCL-2 in its proteolysis of substrates as yet

possibilities

unidentified.

One of the most important

genetically modified at the gene for nCL-2.

questions is how calpain selects specificThis research is now proceeding in our

substrates for proteolysis.

laboratory.

A scaffold forfor

calpaincanbe(30,58).
explored

These
in

mice

proteolysis, like that observed in various

14

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Acknowledgements
We are grateful to Keiji Tanaka, Tomoki Chiba, Shigeo Murata, Choji Taya, and Hiromichi
Yonekawa at Rinshoken, Toshiyuki Takeuchi (Gunma University), Taeko Dohi (International
Medical Center of Japan), Shoichi Ishiura and Ichiro Matsumoto (University of Tokyo), and all of
our laboratory members for experimental support and valuable discussion.

This work was

supported in part by MEXT.KAKENHI 16026209 (to HS), 14704064, 15032201, and 16026201 (toHK), 14086203 (to TM), 14380309 (to KS), and 14656054 (to KA), by JSPS.KAKENHI 15380089

17

(to HS) and 02J07250 (to SH), by a Research Grant (14B-4) for Nervous and Mental Disorders from
the Ministry of Health, Labor and Welfare, and by Ground-Based Research Announcement forSpace Utilization promoted by JSF (to HS).

The abbreviations used are: nCL-2, novel calpain large subunit-2; nCL-4, novel calpain
large subunit-4; COP, coat protein; ISH, in situ hybridization; NP-40, nonidet p-40.

18

FIGURE LEGENDS
Fig. 1. Expression of calpain mRNAs in the mouse stomach oxyntic mucosa. (A) Schematic
structures of the mouse stomach corpus (oxyntic) mucosa.

Serial sections of mouse oxyntic

mucosa were hybridized with Dig-labeled nCL-2 (B), nCL-4 (C), µCL (D), and mCL (E) riboprobes.
The hybridization signals were analyzed by phase contrast microscopy.
with control sense riboprobes (unpublished data).

No signal was observed

Bars, 100 µm.

Fig. 2. Expression of calpain mRNAs in the mouse stomach pyloric mucosa. (A) Schematic
structures of the mouse stomach pyloric mucosa.

Serial sections of mouse pyloric mucosa were

hybridized with Dig-labeled nCL-2 (B), nCL-4 (C), µCL (D), and mCL (E) riboprobes.

Bars, 100µm.
Fig. 3. Specific expression of nCL-2 in the pit cells. (A) Schematic structures of nCL-2 and nCL-2´,and the locations of epitopes for the antibodies used.

(B) Protein (20 µg) from mouse gastricmucosa was analyzed by western blotting probed with anti-nCL2/2´ antibody.
indicates the band corresponding to nCL-2.

The arrowheadnCL-2´ (43 kDa) was not detected.

(C–F) Sectionsof rat stomach oxyntic mucosa were immunostained with anti-nCL2/2´ (C, E, and F) andanti-nCL2C (D) antibodies.
and F).

Signals were visualized by Alexa 594 (C and D) or HRP methods (E(F) A magnified image of the area indicated by the broken square in (E).

(F) indicate nCL-2-negative parietal cells.
+

Arrowheads in(G and H) Rat oxyntic mucosa sections immunostained+with anti-nCL2/2´ (red) and anti-H /K -ATPase (green) antibodies (G), or with anti-nCL2/2´ (red)and anti-PCNA (green) antibodies (H).

Staining with secondary antibody alone yielded negligible

background signal in all experiments (unpublished data).

Bars, 100 µm.

Fig. 4. Expression of nCL-2 in subsets of goblet cells in the duodenum. (A) Northern blot analysis
of various mouse (lanes 1–9) and human (lanes 10–33) tissues using the probes indicated.

Lanes: 1,

testis; 2 and 24, skeletal muscle; 3 and 29, liver; 4 and 28, kidney; 5 and 32, lung; 6, 13, and 22,
brain; 7, large intestine; 8 and 17, stomach; 9 and 30, small intestine; 10, adrenal grand; 11, bladder;
12, bone marrow; 14, lymph node; 15, prostate; 16, spinal cord; 18, thyroid; 19, tongue; 20, trachea;
21, uterus; 23, heart; 25, colon; 26, thymus; 27, spleen; 31, placenta; 33, peripheral blood leukocytes.
(B and C) ISH analysis of mouse duodenum sections was performed using riboprobes for nCL-2 (B)and nCL-4 (C).

The lower two panels are magnified images of the upper panels.

Immunostaining of the rat duodenum using anti-nCL2/2´ antibody.

(D)

Arrows indicate nCL-2

immunoreactive goblet cells characterized by their expanded shape in the apical region.
E, epithelium; LP, lamina propria.

L, lumen;Bars, 100 µm (B and C), 50 µm (D).

19

Fig. 5. Identification of nCL-2-interacting proteins. Schematic structures of nCL-2-binding proteins
identified by yeast two-hybrid screening using domain IV and full-length nCL-2 as baits: Eya2 (A),GPS1 (B), TOM70 (C), β-COP (D).

Thick lines below the schematic structures indicate thepositions and lengths of the identified prey clones (see table 1).

Thin lines in (A) indicate two

truncated constructs used to examine the binding site for Eya2; + and – stand for the binding andunbinding of the fragment to nCL-2, respectively, examined with a yeast two-hybrid system.
closed arrowhead in (D) indicates the proteolytic site of β-COP (see Fig. 7).

The(E) HA-tagged GPS1

(lanes 1 and 2), β-COP (lanes 3 and 4), nm23-M2 (lanes 5 and 6), nm23-M4 (lanes 7 and 8), andEya 2 (lanes 9 and 10) were transfected with FLAG-tagged nCL-2 (odd lanes) or mock vector (evenlanes) into COS7 cells.

The cell lysates were immunoprecipitated with anti-FLAG agarose, andsubjected to western blot analysis using anti-FLAG and anti-HA monoclonal antibodies.

(F) GST(lane 2) or GST-fusion protein (lane 3) of the C-terminal region of TOM70 corresponding to theidentified two-hybrid clone (see (A)) was incubated with FLAG-tagged nCL-2:C105S expressed in
COS7 cells, and pulled-down by glutathione–Sepharose.

Lane 1 shows 10% of the input of

FLAG-nCL-2:C105S.
Fig. 6.

Expression of nCL-2-interacting proteins, GPS1 and β-COP, in mouse stomach pit cells.

β-COP (A and C) and Eya2 (B and D) transcripts were expressed in the pit cells of the mousestomach oxyntic (A and B) and pyloric (C and D) mucosae, demonstrated by ISH.
structure of the stomach section, see Figs. 1 and 2.
Fig. 7.

For theBars, 100 µm.

Proteolytic interaction of β-COP, but not GPS1, nm23-M2, -M4, or Eya2, with nCL-2. (A)HA-tagged GPS1 (lanes 1–4), nm23-M2 (lanes 5–8), nm23-M4 (lanes 9–12), or Eya 2 (lanes 13–16)
were co-transfected into COS7 cells with FLAG-tagged WT or protease-inactive mutant nCL-2 (CS),and the cell lysates were incubated with or without Ca2+ as indicated.
western blot analysis using anti-HA antibody.

Bands were detected by

Closed arrowheads and asterisks indicate thefull-length proteins and non-specific bands, respectively.

(B) HA-tagged β-COP was transfected

into COS7 cells with FLAG-tagged WT (lanes 1–6) or protease-inactive mutant nCL-2 (CS, lanes 7
and 8), and the cell lysates were incubated with (lanes 1 and 3–7) or without (lanes 2 and 8) Ca2+ inthe absence (lanes 1, 2, 7, and 8) or presence of 50 µM E64c (lane 3), 10 µM calpain inhibitor I(lane 4), 10 µM calpeptin (lane 5), or 10 µM MG132 (lane 6).

Bands were detected by western

blot analysis using anti-HA antibody. Closed and open arrowheads indicate full-length β-COP anda proteolytic fragment of β-COP, respectively.

Asterisks indicate non-specific bands.

(C) (upper)

Recombinant β-COP was incubated with or without recombinant nCL-2 in the absence or presenceof Ca2+ as indicated (lane 10 did not contain β-COP), and stained by CBB.

The closed arrow, open

20

and closed arrowheads indicate β-COP, nCL-2, and proteolytic fragments of β-COP, respectively.
Asterisks indicate unrelated proteins co-purified with β-COP.
band was quantified by densitometry.
Fig. 8.

(lower) The intensity of the 62 kDa

Values were means ± SD; n = 3.

Co-localization of nCL-2 with β-COP in the Golgi, and limited proteolysis of β-COP after

Ca2+-ionophore stimulation. (A) COS7 cells transfected with FLAG-tagged nCL-2 (a–l), HA-tagged
β-COP (a–f), or mock vector (m–o) were doubly immunostained with anti-nCL2C (red) and anti-HA
antibodies (green) (a–f), with anti-nCL2C (red) and anti-GM130 (green) antibodies (g–l), or with
anti-β-COP (red) and anti-GM130 antibodies (green) (m–o).

The merged images show

overlapping signals for nCL-2 and β-COP (c and f), nCL-2 and GM130 (i and l), and β-COP and
GM130 (o) in the perinuclear region corresponding to the Golgi.

The cells shown here arerepresentative of the ones most frequently observed.

Bars, 20 µm.

N, nucleus.

(B) COS7 cells transfected with mock vector (lanes 1 and 2), FLAG-tagged nCL-2 (lanes 3 and 4),
or FLAG-tagged nCL-2:C105S (lanes 5 and 6) were treated with dimethylsulfoxide (lanes 1, 3, and5), or 10 µM A23187 (lanes 2, 4, and 6) for 20 hr, and detected by western blot analysis using
anti-β-COP antibody.

An arrow and an arrowhead indicate endogenous β-COP and its proteolytic

fragment, respectively.

(C) Lysate of COS7 cells transfected with FLAG-tagged nCL-2, wasincubated with or without Ca2+, and compared with the sample same as in Fig. 8B lane 4.

Anarrow and an arrowhead indicate endogenous β-COP and its proteolytic fragment, respectively,
detected by western blot analysis using anti-β-COP antibody.
Fig. 9.

The proteolyzed fragment of β-COP localized dispersed from the Golgi.

COS7 cells

transfected with HA-tagged β-COP (full-length), β-COP-N (aa 1–528), or β-COP-C (aa 529–953),
respectively, were doubly immunostained with anti-HA (A, D, and G; red) and anti-GM130 (B, E,
and H; green) antibodies. β-COP-N and -C showed rather diffuse expression patterns that partiallyoverlapped with that of GM130 in the Golgi (C, F, and I).
of the ones most frequently observed.
Fig. 10.

The cells shown here are representative

N, nucleus. Bars, 20 µm.

Schematic illustration of a proposed model for COPI disassembly. A model of thedisassembly of the COPI subcomplex with proteolysis by nCL-2, modified from the model proposedby Hoffman et al. (42).

nCL-2 binds to the ear domain of β-COP through domain IV and also topossible regulatory molecule(s) such as Eya2.

Upon stimulation with Ca2+, nCL-2 proteolyzes

β-COP close to the C-terminal end of the adaptin N-terminal domain to dissociate the polymeric
COPI complex to the single complex.

21

Table 1.

Summary of the results of yeast two-hybrid screening

Count

In vitro Interactiona

nucleotide diphosphate kinase nm23-M2

12+β-subunit of COPI coatomer (β-COP)

2+translocase of outer membrane 70 (TOM70)

2+nucleotide diphosphate kinase nm23-M3

1

n.d.b

eyes absent homologue 2 (Eya2)

1+

G-protein pathway suppressor 1 (GPS1)

1

+

Bait
domain IV

Prey

total
full-length

19vimentin77

n.d.

desmin25

n.d.

nm23-M2

3+

nucleotide diphosphate kinase nm23-M4

3

+

Eya2

2

+

hemopoietic lineage switch protein 5 (Hls5)

2

n.d.

β-COP

1

+

GPS1

1

+

pre-B cell leukemia transcription factor

1

n.d.

hemoglobin α

1

n.d.

axotrophin

1

n.d.

tyrosine kinase-associated leucine zipper protein

1

n.d.

Nedd9

1

n.d.

cardiac actin

1

n.d.

perlican1n.d.

aldo-keto reductase

1

n.d.

RIKEN cDNA clone 3632410O17

1

n.d.

RIKEN cDNA clone 4932411G06

1

n.d.

(unidentified sequence)

18

n.d.

interacting protein 1 (Pbxip1)

142

total
a

For in vitro interactions, see Figs. 5E and F.

bn.d., not determined.

22

Hata et al., Figure 3(A)protease domain

C2-likedomain5EF-handdomainEF-1 2 3 4 5

nCL-2

I

IIa

IIb

Cys

His Asn

III
IV

YYY

anti-nCL2C

nCL-2'

I

IIa

IIb

Cys

His Asn

YYY

anti-nCL2/2'

(B)
(kDa)
175
83
62
47
32

25

anti-nCL2/2'

25

Hata et al., Figure 4(A)(mouse) 1 2 3 4 5 6 7 8 9
nCL-2
nCL-2'mCL
GAPDH
10 11 12 13 14 15 16 17 18 19 20 21(human)nCL-2

22 23 24 25 26 27 28 29 30 31 32 33

27

Hata et al., Figure 5(A)(Eya2)
74(nuc.)

1
1

(a.a)

1669

transactivation
domain2181

Eya-phosphatasedomain

532

63

2181

(3 clones)+(B)(GPS1)(nuc.)

227

1

1642

TPR domain

PCI domain
1

(a.a)

240

408 471

911

(C)

2018

2018

(2 clones)(TOM70)(nuc.)(a.a)

1

Hsp70,90

82
1 TM

1 2

1914

3 4 5 6 7 8 910
TPR domains

5881

611

1204

3710

1231

(D)

2231

(1 clone)(1 clone)(b-COP)(nuc.)
(a.a)

74
1

2935

Adaptin N-terminal domain linker
1

550

650
1748

1511
644

3246

ear domain
953

3246
3246
3246

(1 clone)(1 clone)(1 clone)29

Hata et al., Figure 5 (continued)

(E)

β-COP

nm23-M2

nm23-M4

3

5

7

GPS1
W.B.

1

2

4

6

8

Eya2
9

10

anti-FLAG
anti-HA
anti-FLAG

anti-FLAG

G

G

ST
-T
O

10

%

in

pu

t

(F)

M

70

anti-HAST

I.P:anti-FLAG

1

2

3
nCL-2 C105S

30Hata et al., Fig. 10

C

N

dII

dIII

ζ

α

β'
dIV

γ

nCL-2
(inactive)

N

β
dIII

β'

F-COPI

dII

δ

α

γ

ζ

N

Cytosol

ε

dIV

N

dIII

dII

Golgi

Ca2+
C

C

ε

N

γ
N

dIV

nCL-2
(active)

C

β

δ
ζ

β'

α

β'

ε

dIII

δ
ζ

β
dII

N

α
dIV

γ

C

dIV

β

C

C

regulatory
molecule(s) ?

δ

B-COPI

ε

C

C

dIII

N

dII

CytosolGolgi

35