The importance of conserved amino acid residues in protease sub-domain IIb and the IS2 region in p94 forconstitutive autolysis.

Yasuko Onoa,*, Chikako Hayashia,b, Naoko Doia,c, Mai Tagamia and Hiroyuki Sorimachia,caDepartment of Enzymatic Regulation for Cell Functions (Calpain Project), The Tokyo Metropolitan

Institute of Medical Science (Rinshoken), Tokyo, Japanb

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science,

Chiba, Japanc

CREST, Japan Science and Technology (JST), Saitama, Japan*Corresponding author: Yasuko Ono, Department of Enzymatic Regulation for Cell Functions (Calpain
Project), The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-3823-2182; Fax: 81-3-3823-2359; E-mail:yakoono@rinshoken.or.jp

1Abstractp94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized byapparent Ca2+-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates.
The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in
the cytosol without an extra Ca2+. Ca2+-dependent p94 mutants were screened using “p94-trapping”, which
is an application of yeast genetic reporter system called “proteinase-trapping”. Several amino acids wererevealed as critical for apparent Ca2+-independent p94 activity. These results highlight the importance ofconserved amino acids in domain IIb as well as in the p94-specific IS2 region.

Keywords: calpain; p94/calpain 3; autolytic activity; protease activity; Ca2+-dependence

Abbreviations: AD, activation domain of Gal4; BD, DNA binding domain of Gal4; LGMD2A, limb-girdle
muscular dystrophy type 2A; Sf-9, Spodoptera frugiperda cells; WT, wild type; CS, C129S protease
deficient mutation; DA, D607A missense mutation; IS1 and IS2, p94-specific insertion sequence 1 and 2,
respectively; NS, N-terminal sequence unique to p94; CAST-d1, calpastatin domain 1; SD-WHA, synthetic
minimal medium with dextrose minus tryptophan, histidine and adenine.

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1. Introduction
Calpains (EC 3.4.22.18, clan CA) comprise a unique branch of the cysteine protease superfamily [1-3].
Studies on μ- and m-calpains, or the “conventional” calpains, have shown the importance of Ca2+ inregulating calpain activity. Crystal structures of calpain identified Ca2+-binding sites in proteasesub-domains IIa and IIb as well as domain IV composed of 5 EF-hand motifs [4-6]. However, there areseveral calpain paralogues to which the relevance of Ca2+ is not clear, as represented by p94/calpain 3, theskeletal muscle-specific member of the calpain family.
The nature of p94 protease activity is largely unknown except that p94 has very rapid and exhaustive
autolytic activity that is apparently Ca2+-independent. Further, the interaction of p94 with sarcomeric
proteins, such as connectin/titin, is considered one of the in vivo mechanisms for regulating p94 localizationand stability [7-11]. The importance of p94 function in human skeletal muscle is suggested because
mutations in the p94 gene cause limb-girdle muscular dystrophy type 2A (LGMD2A) [12,13]. Importantly,
although autolytic activity of p94 demonstrates protease activity, proteolytic activity against non-self
substrates would be expected to be most physiologically important [13].
The aim of this study is to clarify the molecular basis for Ca2+-independent p94 protease activity. The
yeast “proteinase-trapping system” [14] was modified to screen the p94 missense mutants whose
constitutive autolytic activity were transformed to be explicitly Ca2+-dependent [10]. Two groups of aminoacids were shown to be essential for the apparent Ca2+-independence of p94 protease activity. One iscomposed of conserved amino acid residues within IS2, and the another is in domain IIb proximal to those
involved in Ca2+-binding [6].

2. Materials and Methods
2.1. General techniques
Standard protocols for manipulating yeast cells, and expression of recombinant proteins in COS7 cells
and Spodoptera frugiperda (Sf-9) cells were described previously [15]. For detecting p94 expression,
COS7 cells were harvested 72 h after electroporation, and sonicated in buffer A (20 mM Tris-HCl, pH 7.5,
1 mM EDTA, 1 mM DTT, and 2 mM PMSF). The supernatant obtained by centrifugation at 1,470×g for 5
min at 4oC was immediately mixed with Laemmli SDS-PAGE sample buffer followed by heat denaturing at
95oC for 3 min. For the proteins expressed in Sf-9 cells, essentially the same procedures were used except

3

for centrifugation at 20,630×g for 30 min at 4oC. Antibodies used for western blotting includeanti-p94-pIS2 [10], anti-p94-pNS [7,10], and RP4CALPAIN3 antibodies (Triple Point Biologics).

2.2. cDNA constructs
Enzymes were purchased from TAKARA BIO, Stratagene, or New England Biolabs. The expression
vectors were constructed using human p94/calpain 3 cDNA, pSRD, pFastBac1 (Invitrogen) and pAS2-1C
as previously described [10]. Mutations were introduced by PCR using Pfu, Pfu-Turbo, or ExTaq DNA
polymerases, and all DNA sequences were verified [10].

2.3. Screening for p94 mutants defective in autolytic activity
Random mutations were introduced into the sequence for IIb-III-IS2-IV (aar 342-821 of NP_000061) of
p94 using primers, 5´-GATGAGGTCCCGTTCAAAGG-3´ and 5´-GGCAATTGGTTGTGAAAGCGC-3´,
as described previously [10]. The PCR products (20 μg) were cotransformed with 20 μg of the 9.3 kb
NotI–Tth111I fragment of the BD–p94–AD expression vector into Saccharomyces cerevisiae AH109 tocause in vivo homologous recombination (Fig. 2C). Transformants selected on the plates,
SD–Trp/–His/–Ade (SD–WHA), were further characterized as to their Ca2+/Na+-sensitivities using theplates supplemented with 0.1M or 0.2M CaCl2 (SD–WHA/Ca) or 0.2M NaCl (SD–WHA/Na). Plasmid
DNA was isolated from selected colonies, and mutations were identified by DNA sequencing.

2.4. Protease activity assay
Protease activity of p94 was analyzed using the supernatant of Sf-9 cells prepared as described above.
For proteolysis of calpastatin, final protein concentrations of cell lysate and calpastatin(CAST)-domain 1(d1) (TAKARA BIO) were 0.8 μg/μl and 0.1 μg/μl (ca.7 μM), respectively, which causes inhibition of theconventional calpains. The reaction was terminated at the indicated times and was analyzed by SDS-PAGE.

3. Results
3.1. Characterization of p94:D607A mutant protease activity
Studies on p94 splicing variants, such as Lp82 and p94Δ [15-18], have implicated that a portion of theIS2 region encoded by an exon 16 is involved in apparently Ca2+-independent rapid autolysis of p94 (Fig.

41A). Accordingly, a short stretch of amino acids in IS2 is highly conserved among p94 from various species.
Since acidic amino acids are important for Ca2+-binding, expression of p94:D607A(DA) and p94:E614A in
COS7 cells were examined. Both mutants presented Ca2+-dependent autolytic activity, and, p94:DA wasslightly more stable than p94:E614A (data not shown).
Therefore, p94:DA was expressed in Sf-9 cells for further studies. p94:WT was detected exclusively as
autolyzed fragments (Fig. 1B, lane 1). In contrast, the full-length fragment and autolytic fragments weredetected for p94:DA (Fig. 1B, lane 2). Reactivities to the antibodies suggested that p94:DA lacks the
N-terminal region of NS, possibly because of autolysis after Ala14 (Fig. 1A, closed arrowhead 1, and Fig.
3B, lane 1). Autolytic activity of p94:DA was Ca2+-dependent (Fig. 1B, lanes 2-5). Under the same
conditions, the protease inactive mutant, p94:C129S(CS), demonstrated no autolysis (Fig. 1B, lanes 6-8).
The protease activity of p94:DA against a non-self substrate was examined using d1 of CAST, a specificinhibitor of conventional calpains and a good substrate for p94:WT. The observed MW for CAST-d1 (22kDa) was higher than calculated (14.2 kDa) (Fig. 1C, arrow). In the presence of Ca2+, p94:DA very
efficiently proteolyzed CAST-d1, and CAST-d1 did not inhibit the autolysis of p94:DA (Fig. 1C, lanes 1-5).
These data demonstrate that p94:DA is the first designed missense mutant that recapitulates, albeit
Ca2+-dependently, some characteristics of p94:WT protease activity.

3.2. Detection of Ca2+-dependent autolytic activity of p94:DA in “p94-trapping”
In yeast “p94-trapping”, p94 autolytic activity reduces transcriptional activation ability of Gal4-p94
hybrid protein, BD-p94-AD, resulting in less growth of yeast cells in selection medium [10] (Fig. 2A,
SD-WHA, WT vs CS).
The yeast cells expressing BD-p94:DA-AD was comparable to those expressing BD-p94:CS-AD as to
the growth on SD-WHA plates. When the plates were supplemented with either 0.1 M CaCl2 or 0.2 M NaCl,
however, the cells expressing BD-p94:DA-AD failed to grow (Fig. 2A, SD-WHA with CaCl2 or NaCl).
Exposure of yeast cells to high [NaCl], but not [KCl], causes transient increase of [Ca2+]i by a mechanism
for cellular ionic homeostasis [19,20]. Accordingly, KCl was not significant (Fig. 1C, SD-WHA, KCl).
These observations indicate that “p94-trapping” is useful to discriminate conditionally active p94
mutants like p94:DA from p94:WT or p94:CS based on Ca2+/Na+-sensitivity of yeasts (Fig. 2B).

5

3.3. Isolation of p94 mutants with Ca2+/Na+-dependent autolytic activity
p94 mutants with autolytic activity similar to that of p94:DA were screened in “p94-trapping” using thesensitivity to Ca2+/Na+ of yeasts as an indicator. Random mutations were introduced into IIb-III-IS2-IV
domains of BD-p94:WT-AD by PCR and by in vivo homologous recombination (Fig. 2C). Since theexpression of tandemly linked BD/AD domains is a prerequisite for yeast to grow on SD-WHA plates,
nonsense mutations in p94 are excluded, which is advantageous for isolating missense mutants of interest.
Out of 2,796 yeast colonies viable on SD-WHA (ca. 2.5x105 transformants screened), 26 colonies wereidentified as Ca2+/Na+-sensitive. Finally, 18 different p94 mutants were identified in 19 colonies (Table 1,
Fig. 2D). Selected plasmids (Table 1, No. 6, 9, 14, 15, 17, and 18) were re-introduced into yeasts to confirm
that Ca2+/Na+-sensitivity of the colonies were dependent on the expression of Gal4-p94 mutant proteins.
The results indicated that F604 and V605 as well as rationally anticipated D607 were relevant to p94
autolytic activity (Table 1, mutants 15-18). Another intriguing finding is that mutations found in domain IIb
are adjacent to theoretical Ca2+-binding residues [6,21]. This further suggests that protease domain of p94 is
activated by a mechanism similar to that of the conventional calpains, and that other regions of p94
facilitate the process spontaneously, i.e., in an apparently Ca2+-independent mechanism [22].
For the clones having two mutations (Table 1, No. 5, 9, 10, 13, and 14), F604L, S423P, V431E, N539D,
and I603T are considered primarily responsible, although cooperative effect of two mutations is also
possible. The rationale is as follows: (i) only significantly weakened autolytic activity of p94 mutants lets
yeasts grow on SD-WHA. Therefore, mutations without suppressive effect on p94 autolysis in COS7 cells,
V590M and Y770H, were excluded (Fig. 3A, lanes 4 and 7); (ii) F604L or F604S, respectively, wasidentified as a responsible single mutation in clone 15 or 16; (iii) mutations in domain IV of p94 tend toenhance p94 autolysis as is shown for Y770H (Fig. 3A, lane 7), S744G and R769Q [13], suggesting thesame trend for M792T and S739P; (v) N539 is highly conserved in the calpain superfamily; (iv) I603 is oneof well-conserved amino acids encoded by exon 16 (Fig. 1A).
Consistent with above, reduced, but not zero, autolytic activity was observed when p94:S423P or
p94:D607N was expressed in COS7 cells (Fig. 3A, lane 3). Both mutants exhibited Ca2+-dependent
autolytic activity comparable to that of p94:DA (data not shown).

3.4. Difference among mutations in domain IIb and IS2

6

The impact of mutations identified in domain IIb and IS2 on enzymatic capability of p94 were further
analyzed using p94:E393K, p94:F604L, and p94:W360Y. W360 corresponds to W298 and W288 in μCL
and mCL, respectively, which undergo a transposition upon activation, thus important for activity [6,23].
The specific activity of m-calpain:W288Y is only 5 % of that of wild type [24], and, consistently, themutation W360C in p94 is associated with LGMD2A [25].
When expressed in Sf-9 cells, autolysis of p94:W360Y was even more retarded than other mutants were,generating an intermediate autolyzed fragment (Fig. 3B, anti-pIS2, grey arrowhead). Antibody reactivities
showed that p94:W360Y retained the intact N-terminus as p94:CS did while other mutants underwent
N-terminal autolysis as p94:DA did (Fig. 1A, closed arrowhead 1, Fig. 3B, anti-pNS, closed arrowhead).
All the mutants proteolyzed calpastatin (Fig. 3C, arrows), and autolyzed Ca2+-dependently even withcalpastatin (Fig. 3C, closed arrowheads), suggesting that some of p94 unique properties other than apparent
Ca2+-independency are intact in these mutants. A preference for autolysis or proteolysis of calpastatin was
different for each mutant as follows: p94:F604L was almost identical to p94:DA (Fig. 3C, lanes 13 to 15 vsFig. 1C, lanes 3 to 5). p94:E393K was less efficient than p94:DA in proteolyzing calpastatin, however, it
exhibited most exhaustive autolytic activity among the mutants examined (Fig. 3C, lanes 8 to 10). On theother hand, both activities were significantly retarded in p94:W360Y (Fig. 3C, lanes 3 to 5).

4. Discussion
This study demonstrated that a “p94-trapping” system, a yeast genetic reporter system for monitoring
p94 autolytic activity, provided an effective method to screen p94 mutants for conditional, i.e.,
Ca2+-dependent, autolytic activity. We anticipated that identified mutations suggest amino acids allowing
p94 to hold an active conformation within the cellular environment. Some mutations identified in our
screening are localized in the same or flanking positions of LGMD2A mutations (Table I). This exemplifies
that mutations transforming p94 protease activity to be Ca2+-dependent are not functional in vivo. In otherwords, apparently Ca2+-independent activation of p94 is in fact physiologically essential.
The p94:DA and p94:F604L mutants clearly indicated that the IS2 region is responsible for activating
p94 in the absence or in the presence of very low level of Ca2+. It is also possible that mutated amino acidpositions within domains IIb to III are important for receiving and filtering changes in IS2 to the whole
molecule. In this regard, five of the six identified mutations in domain IIb (V363A, W373R, V379E, L387P,

7

and E393K; see Figs. 2D and 4A) are adjacent, but not identical, to the Ca2+-chelating residues in μCL and
mCL, while Q390 corresponds to V327/R317 in μCL/mCL, whose backbone nitrogen is used forstabilization of a water molecule involved in Ca2+-coordination [6]. Furthermore, all of six residues, at leastin part, exist on the surface of the molecule (Fig. 4B) as has been shown for LGMD2A-associatedmutations projected to the crystal structure of m-calpain [26]. These results suggest that the domain IIb
mutations, including Q390P, very mildly affect the structure of the Ca2+-binding site of p94, and/or thatthese residues interact with IS2. Further crystallographic studies on the structure of full-length p94 will
elucidate the mechanisms how the IS2 region alters the structure of p94, especially the projection of theamino acids conserved between p94 and the conventional calpains, and how autolysis and substrate
recognition are coordinated within p94.
The primary advantage of conditionally active p94 missense mutants over spliced variants of p94, suchas Lp82 and p94Δ [15,17], as alternatives for p94:WT is that these mutants are expected to retain almost
intact p94 structure except for a very local distortion. Identification of p94 mutants using “p94-trapping”
under appropriate screening conditions (such as screening with temperature shifts, cDNA libraries,
chemicals, etc.) might show how p94 autolytic products interact with cellular components, which appears
to be impossible with p94:WT. Studying these p94 mutants would contribute to revealing the regulatory
mechanisms of calpain in general and isoform-specific contexts.

Acknowledgements: We would like to express our gratitude to all “Calpain Project” members forexperimental support and valuable discussions and Katsuhide Yoshioka and Dr. Tatsuya Maeda for their
help in establishing “p94-trapping”. This work was supported in part by research grants MEXT.KAKENHI
17028055 and 18076007 (to H.S.), JSPS.KAKENHI 18770124 (to Y.O.), 18380085 (to H.S.), Research
Grant 17A-10 for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare (to H.S.),
and the Takeda Science Foundation research grant (to H.S.).

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Figure legends
Figure1. Characterization of p94:DA mutant. (A) Schematic structure of p94. Amino acid sequences for theIS2 region encoded by exons 15 and 16 are shown below. Shaded residues are conserved in all 4 species.
Closed arrowheads 1-5, reported autolytic sites. Horizontal bars, the regions used as antigens. (B) Autolytic
activity of p94:DA. p94:WT, p94:DA or p94:CS were expressed in Sf-9 cells. p94:DA has weak autolytic
activity which is enhanced by CaCl2. Closed and open arrowheads, pre-autolyzed and autolyzed fragmentsof p94, respectively. (C) Proteolysis of calpastatin by p94:DA. In upper gels visualized by CBB-staining,
Ca2+-dependent proteolysis of calpastatin domain 1 (CAST-d1) (arrow) was observed when incubated withthe lysates of Sf-9 cells expressing p94:DA (lanes 3-5), but not p94:CS (lanes 7-9). Closed arrowhead, thefull-length product of p94:DA and p94:CS. Autolytic activity of p94:DA was resistant to CAST-d1.

Figure 2. Screening of conditional p94 mutants in the p94-trapping system. (A) Phenotype of p94:DA in the
p94-trapping system. Growth of yeast cells expressing Gal4-p94 hybrid protein (BD-p94-AD) for p94:WT,
p94:DA, or p94:CS after 72 h at 30oC on selection medium plates with supplement as indicated. Autolytic
activity results in less growth. D607A caused poor growth on SD-WHA plates supplemented with CaCl2 or
NaCl. (B) Relationships of autolytic activity of p94 profiled in vitro and in yeast cells. Autolysis of p94:DA(see Fig. 1B) and growth phenotype of yeast cells expressing BD-p94:DA-AD (see Fig.1C), aresummarized. (C) Principle of isolating conditionally active p94 mutants by random mutagenesis in“p94-trapping”. The growth defect on selection medium supplemented with an excess amount of CaCl2 or
NaCl is anticipated to reflect the autolytic degradation of Gal4-p94 hybrid protein induced by an increaseof [Ca2+]i and/or [Na+]i in yeast cells. (D) A schematic view of missense mutations of p94 identified by thescreening. The positions of mutations are categorized into five groups, highly conserved (top) to diverged(bottom). Arrows and *, respectively, stand for conserved and non-conserved amino acid positions
corresponding to those involved in Ca2+-binding of the conventional calpain protease domain.

11

Figure3. Difference among p94 mutants in autolysis and substrate proteolysis. (A) Expression of selectedmutants of p94 in COS7 cells. For p94:S423P and p94:D607N identified by the screening, thepre-autolyzed fragment (closed arrowhead) as well as autolyzed fragments (open arrowheads) weredetected as for p94:DA. p94:V590M and p94:Y770H were detected merely as autolyzed fragments as in
the case for p94:WT. (B) Heterogeneity of autolysis in NS and IS1 among p94 mutants expressed in Sf-9
cells. The largest fragments (closed arrowhead) for p94:DA, E393K, and F604L lost the reactivity to
anti-pNS, whereas those for p94:CS and W360Y retained it. Judging from the size, p94:DA, E393K, andF604L probably autolyzed at the C-terminus of Ala14. Open and gray arrowheads indicate autolyzed
fragments generated by the cleavage in IS1 as indicated by closed arrowheads 3 or 4 and 5 in Fig. 1A,
respectively. *, non-specific signals. (C) Difference in autolytic and proteolytic activities against CAST-d1
among Ca2+-dependent p94 mutants. The upper and lower images show CAST-d1 proteolysis and p94
autolysis, respectively. The mutation W360Y retarded both p94 autolysis and CAST-d1 proteolysis (lanes 3to 5), while p94:E393K expressed autolytic activity more preferentially than proteolytic activity against
CAST-d1 (lanes 8-10). p94:F604L presented essentially the same trend as p94:DA (lanes 13-15).

Figure 4. Three-dimensional modeling of domain IIb with mutated amino acids. The stereo view models of
domain IIb of the rat μ-calpain in the presence of Ca2+ (from 1KXR) were drawn in solid ribbon (A) andsurface (B) schemes using WebLab Viewer Pro Ver. 3.5 (Molecular Simulations Inc.). (A) Active site
residues, Ca2+-binding residues, and the mutated residues indentified in this study are indicated by ball andstick representation in red, blue, and yellow, respectively. The residue shown in black is involved in
Ca2+-binding and also identified as mutated in this study. (B) Mutated residues are indicated in yellow.
Residue numbers shown in (A) correspond to those for human p94 (NP_000061).

12

Table 1

Summary of p94 mutants expressed as BD-p94-AD in colonies with Ca2+/Na+ sensitivity
Mutated amino acids were categorized as follows (see Fig.2D); conserved among calpain superfamily (A),mammalian calpains (B), mammalian typical calpains (C), p94 species (P), or diverged (D). Linkers 1 and
2, respectively, represent regions between domains IIb and III and between III and IS2. For double mutants,
mutations predicted to be primarily responsible for the phenotype are shown in bold face.
No.

MutantDomain(s)

Rank

1

V363A

IIb

C

1

2

W373R

IIb

D

2

3

V379E

IIb

C

1

4

L387P

IIb

B

1

5

Q390P/F604L

IIb/IS2

D/P

1

6

E393K

IIb

B

1

7

T417A

linker1

B

1

T417M

8

L421P

linker1

B

1(A420D)9

S423P/Y770H

linker1/IV

C/B

1(R769Q)

10

V431E/M792T

III/IV

C/C

1

V431M, (E791S)

11

L481Q

III

B

1

12

H519L

III

B

1

13

N539D/S739P

III/IV

A/C

1

14

V590M/I603T

linker2/IS2

D/P

1

15

F604L

IS2

P

1

16

F604S

IS2

P

1

17

V605A

IS2

P

1

(S606L)

18

D607N

IS2

P

1

(S606L)

Total

Number of
independent
clonesidentified

19

13

LGMD2A
mutation in thesame (flanking)sites(R386C)Figure 1.

A

Cys129RP4 calp3
anti-pNS

His334 Asn358
anti-pIS2

IS1 IIb

IIa

NS
12

III

IV

IS2

345

Asp607: P20807
human: Q64691
mouse: Q92177
chickenfugu p94-1 : SINFRUP00000174744

600
*

610
*

620
*

ex15

ex16

B
p94:WT

p94:DA

p94:CS[EDTA][EDTA] [CaCl 2 ][EDTA] [CaCl 2 ]

2

2

2

5

1

2

2

5

1

2

3

4

5

6

7

8

0

17.5

kDa
97

kDa

kDa

97

97

66

66

45

45

(mM)

66
45

30
30

0

30

0

17.5

hr at 4 o C

anti-pIS2C[EDTA] [CaCl2]
5 mM
2 mM
0 90 30 60 90
1

2

3

630
*

KKKKTKPIIFVSDRANSNKELGVDQESEEGKGKTSPDKQKQSPQKKKKNKPIIFVSDRANSNKELGVDQEAEEGKDKAGPEKRGETPQKKKKGKPIIFVSDRANSNKELTTDEDAGKDGEKTHVDEKKRSSASVIVPLRPCQPIVFVSDRARANKE IEHD GIRGEKRKPKGRFSSFV4[EDTA] [CaCl2]
2 mM
5 mM
0 30 60 90 min at 37oC

5

6

kDa

kDa

20

20

97

97

66

66

45

45

30

30

p94:DA

7

8

9

CAST-d1
CBB

anti-pIS2

p94:CS

Figure 2.
A
SD-W

SD-WHA
CaCl2 0.1 M

BD-p94-AD

KCl 0.2 M

NaCl 0.2 M

WT
CS
DA
104 103 102 101
No. of yeast cells

BC
autolysis

IV

WT

CS

DA

EDTA

N/A

-

-

CaCl 2

N/A

-

+

BD NS

C

HN

IIa

IS1 IIb

III

IS2

AD

Tth111I

SD-WHA-++/ CaCl 2-+-/ NaCl

-

+

-

/ KCl

-

+

+

growth ofyeast cells
expressing
BD-p94-ADIIb

in vivo homologous
recombinationin yeast AH109

NotI

pAS2-1C

selection by
Ca2+ or Na+ sensitivity

SD-WHA

SD-WHAw/ Ca2+ or Na++-

ViabilityX

BD-p94-AD w/ mutation(s)

Y
IV

BD NS

IIa

IS1 IIb

III

IS2

plasmid rescue
for sequencing
pAS2-1CµCL E302
mCL E292

D309
D299

V327 M329
R317 H319

D331
D321

E333
E323

E364

D371

Q390 T392

D394

E396

**

III

Y770H
M792T

S739P
I603T
F604L/S
V605A
D607N
V590M

Q390P

p94

Diverged

IV

H519L

L481Q
S423P
V431E

V379E

mammaliantypical calpains

V363A

mammaliancalpains

E393K
T417A
L421P

L387P

calpain super family

Conserved
among:

IS2
N539D

IIb

W373R

D

AD

Y7
7

CS

7

8

1

2

3

T

kDa
97

5

0H

N
07
D6

6

-

S4
2

4

p94:

W

A

3P
V5
90
M
DA

Figure 3.

66
45

30

anti-pIS2

B
0Y 3K 4L
36 39 60 S
A
D W E
F C

1

kDa

2

3

4

5

97
66
45

30

anti-pIS2

RP4 CALPAIN3*
anti-pNSC[EDTA] [CaCl2]
2 mM
5 mM
0 90 30 60 90[EDTA] [CaCl2]
2 mM
5 mM
0 90 30 60 90
1

2

3

6

4 5

7

8

[EDTA] [CaCl2]
2 mM
5 mM
o
0 90 30 60 90 min at 37 C

9 10

11 12 13 14 15

kDa
20

kDa
20

kDa
20

97
66

97
66

97
66

45

45

45

30

30

30

p94:W360Y

p94:E393K

CAST-d1
CBB

anti-pIS2

p94:F604L

Figure 4.

A
T392

Q390P
L387P

D394
E396

N358
H334

E393K

V363A

W373R

E364
W365

B

V379E

D371