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Yeast 15, 1437–1448 (1999)
The Proteolytic System of the Yeast
Kluyveromyces lactis
MARIuA V. FLORES1, ANAHIu CUELLAS1 AND CLAUDIO E. VOGET2*
1
Departamento de Ciencia y Tecnologı́a, Universidad Nacional de Quilmes, Roque Saenz Peña 180 (1876) Bernal,
Argentina
2
Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, CONICET, 47 y 115 (1900), La Plata, Argentina
Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This
yeast expressed proteolytic activities similar to those found in S. cerevisiae. This fact was particularly evident in the
case of proteases such as PrA, PrB and CpY with regard to substrate specificity, activation at pH 5·0 and inhibition
patterns. The presence of a CpS activity could not be detected in either fresh or activated cell-free extracts by using
the dipeptide N-Cbz-Gly-Leu, even in the presence of Zn +2. On the other hand, K. lactis exhibits at least two major
intracellular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely
related proteins. These activities corresponded to molecular masses of about 60 kDa, close pI values, and a similar
behaviour in non-denaturing polyacrylamide electrophoresis. Both activities were enhanced by Co +2 and inhibited
by EDTA. Among different aminoacyl-p-NAs, they preferentially hydrolysed Lys-p-NA. No increase of Ap activity
was obtained by incubation of extracts at acid pH. The maximum PrA and PrB activities detected in N-limited
cultures were six-fold higher than those expressed under C- or P-limitation. The effect of culture conditions on the
Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being
slightly higher in C-limited cells. This fact suggests that hydrolysis of protein to peptides might be the limiting step
in the pathway of general protein degradation in the vacuole. Copyright 1999 John Wiley & Sons, Ltd.
  — Kluyveromyces lactis; proteolytic system; chemostat cultures
INTRODUCTION
Intracellular proteolytic activities in yeast have
been actively studied in the last decades, and their
vital importance in cellular control has become
apparent. Most of the research on proteases of
yeast has been carried out in S. cerevisiae, in which
numerous proteolytic enzymes have been found
and several of them have been biochemically
and genetically characterized (Hirsh et al., 1989b;
Jones, 1991; Bart Van Den Hazel et al., 1996).
There are basically three groups of proteases:
vacuolar proteases; the cytosolic proteosome; and
proteases located along the secretory pathway.
Major proteolytic activities, such as endoproteinase yscA (PrA), endoproteinase yscB (PrB),
carboxypeptidase yscY (CpY), carboxypeptidase
yscS (CpS), aminopeptidase yscI (ApI), amino*Correspondence to: C. E. Voget, CINDEFI, Facultad de
Ciencias Exactas, UNLP, 47 y 115 (1900), La Plata. Argentina.
Fax/Tel: 54 21 833794; e-mail: voget@biol.unlp.edu.ar
CCC 0749–503X/99/141437–12$17.50
Copyright 1999 John Wiley & Sons, Ltd.
peptidase yscCo (ApCo), aminopeptidase yscY
(ApY) (which might be the precursor of ApCo)
and dipeptidyl aminopeptidase yscB (DPAP-B),
were found in the vacuole. These proteases are
involved in processes of cell differentiation,
maturation of precursors and activation of zymogens of the vacuolar proteases (this function is
mainly dependent on PrA activity), general protein
degradation (particularly under nutritional stress),
metabolism of exogenously supplied peptides, and
other functions. The cytoplasmatic proteasome
(multicatalytic proteinase system) is responsible
for a selective, rapid degradation of short-lived
proteins, including proteins that are detrimental
to the cell. The interplay between vacuolar and
proteosome proteolysis is a key regulatory cell
process, particularly in response to stress (Teichert
et al., 1989; Hilt and Wolf, 1992; Lee and
Goldberg, 1996; Bart Van Den Hazel et al., 1996).
The proteases of the secretory pathway, which
are mainly located in the Golgi apparatus and
Received 7 January 1999
Accepted 22 March 1999
1438
the plasmatic membrane, act to process
precursors to one or more secreted peptides.
Kluyveromyces lactis has elicited both an
academic interest as a genetic model and industrial
interest as a source of diverse metabolites, enzymes
such as â-galactosidase, and also as an attractive
microbial host for the expression of foreign genes
and protein secretion (Romanos et al., 1992;
Bonekamp and Oosterom, 1994). Characterization of endogenous proteases is useful in
designing processes for the production of yeast
autolysates, to stabilize cells for biotransformations, and to define strategies for protein purification. In addition, the use of yeast for molecular
cloning requires precise knowledge of its proteolytic system, because endogenous proteases may
participate in the post-translational maturation
and/or degradation of a product of a newly-cloned
gene.
There is almost no information on the proteolytic activities of K. lactis, except for some reports
on a preliminary identification of some endoproteinases and carboxypeptidases (Grieve et al.,
1983). The present paper reports a description of
the major intracellular proteinase activities present
in the yeast K. lactis, strain NRRL 1118. This
strain had been previously used to study the physiology of growth and â-galactosidase synthesis in
aerobic chemostat cultures (Inchaurrondo et al.,
1998).
MATERIALS AND METHODS
Substrates and chemicals
Azocoll, haemoglobin, aminoacyl-p-nitroanilides
(A-p-NA), aminoacyl-â-naphthylamides (A-âNA), glycyl--proline-p-nitroanilide (Gly-Pro-pNA), N-benzoyl--tyrosine-p-nitroanilide (BTPNA),
N-carbobenzyloxy-glycyl-leucine (N-CBZ-Gly-Leu),
N-carbobenzyloxy-phenylalanyl-leucine (N-CBZPhe-Leu), Fast Garnet, PMSF, pepstatin, lyticase
(from Arthrobacter luteus, L 5763), ONPG and
ninhydrin reagent solution were purchased from
Sigma (St Louis, MO). Other chemicals were of the
best analytical grade. Equipment and chemicals for
chromatography and electrophoresis procedures
were obtained from Pharmacia Biotech.
Yeast strain and culture conditions
The yeast strain used was K. lactis NRRL
Y1118 (ARS Culture Collection, Peoria, IL,
USA). Yeast was grown in aerobic chemostat
Copyright 1999 John Wiley & Sons, Ltd.
M. V. FLORES ET AL.
cultures under carbon (lactose), nitrogen (NH3) or
phosphate limitation at a growth rate of 0·1 h1
using a synthetic medium. Medium composition
and growth conditions have been reported elsewhere (Inchaurrondo et al., 1998). Biomass from
the culture media was recovered by centrifugation
at 5C (5000 g10 min), washed twice with distilled water and frozen at 20C until used. In
some experiments, fresh cells were used instead of
frozen and thawed cells.
Preparation of cell-free extracts
Frozen cells were thawed and suspended in 0·1 
potassium phosphate buffer, pH 7·0, at a cell concentration of 1·0–1·51010 cells/ml (1 mg cell dry
weight 2108 cells). The suspension was mixed
with glass beads (0·45–0·50 ìm) and cells were
broken in a cell homogenizer (MSK, B Braun,
Melsungen). The crude homogenate was centrifuged at 20 000g for 20 min at 5C. The turbid
supernatant fluid (S20 000) was removed and centrifuged again for 2 h at 100 000g at 5C using a
Beckman ultracentrifuge. The corresponding
supernatant (S100 000) was used as soluble extract.
The pellet (P100 000 g) was resuspended in the
same buffer and used for enzymatic analysis. To
activate the proteolytic enzymes, the pH of the
soluble extract was adjusted to pH 5·0 by adding
glacial acetic acid with continuous mixing. The
precipitate formed was separated by centrifugation
(the protein precipitated as result of acidification
was around 20–35% of its original concentration in
the fresh extract). Thereafter, the clear supernatant
was incubated at 25C. Sodium azide (0·02%)
was added as a preservative. Samples for analysis
were taken at selected time intervals, mixed with
glycerol (10% w/w) and frozen for analysis. Activation of proteases was achieved by digestion of
proteinase inhibitors (Hirsh et al., 1989b).
Enzyme assays
Proteinase A (PrA) activity was assayed with
haemoglobin as substrate (Meussdoerffer et al.,
1980). A sample (0·1 ml) was incubated with 0·4 ml
of 1·2% acid-denatured haemoglobin, buffered at
pH 3·4 with KOH, for 30–60 min. The digestion
was stopped by adding 0·5 ml of 10% TCA. After
10 min, the sample was centrifuged in a microfuge
(10 000g for 10 min). The split products in the
supernatant were measured by the Lowry method
(Lowry et al., 1951), using tyrosine as standard.
Yeast 15, 1437–1448 (1999)
PROTEOLYTIC SYSTEM OF K. LACTIS
One unit of PrA corresponded to the amount of
enzyme, which gives an absorbance at 660 nm
equivalent to 1 ìg of tyrosine in 1 min.
Proteinase B (PrB) activity was assayed with
Azocoll as substrate (Saheki and Holzer, 1974).
The reaction was carried out in 1·5 ml microfuge
tubes containing 12 mg of Azocoll, 0·4 ml 0·1 
potassium phosphate buffer (pH 7·0), and 0·1 ml
sample. After an appropriate reaction time
(usually 60–90 min), the reaction was stopped by
adding 0·5 ml 10% TCA. After 10 min, samples
were centrifuged (10 000g for 10 min) and the
absorbance of the supernatant measured at
520 nm. One unit of PrB corresponded to the
amount of enzyme, which gave an increase
in absorbance of 1·0 unit/min in the supernatant
solution.
Carboxypeptidase (Cp) activity was assayed
with BTPNA and with the dipeptides N-Cbz-GlyLeu and N-Cbz-Phe-Leu (Aibara et al., 1971;
Hayashi et al., 1973). The assay with BTPNA was
carried out by incubating 50 ìl sample with 1 ml
0·6 m substrate in 0·1  potassium phosphate
buffer, pH 7·0, containing 0·2% sodium deoxycholate and 10% N,N-dimethylformamide. The
BTPNA stock solution (30 m) was prepared in
N,N-dimethylformamide. Activity with N-CbzGly-Leu or N-Cbz-Phe-Leu was determined by
measuring the release of leucine with the ninhydrin
reagent (Sarath et al., 1981). One ml of substrate
solution (5 m N-CBZ-Gly-Leu or N-CBZ-PheLeu in 0·1  MOPS, pH 7·0) was incubated with
50–100 ìl sample. At intervals, a 200 ìl portion of
the assay mixture was removed, mixed with 1 ml
ninhydrin solution (10% ninhydrin reagent in 0·5 
sodium citrate buffer, pH 5·0) and heated in boiling water for 15 min. After cooling, the absorbance of the solution was determined at 570 nm.
-leucine was used as standard. One unit of
carboxypeptidase activity corresponded to the
amount of enzyme that liberated 1 ìmol leucine or
released 1 ìmol p-nitroanilide/min.
Aminopeptidase (Ap) activity was assayed
with aminoacyl-p-nitroanilides. Unless otherwise
stated, the assay was done by mixing 20–50 ìl
sample with 900 ìl 50 m Tris–HCl, pH 7·2. After
5 min, the reaction was started by adding 100 ìl of
A-p-NA stock solution (10 m in 5 m H2SO4).
Ap was also determined with aminoacyl-ânaphthylamides. In this case, the A-â-NA substrate was directly prepared in buffer. Dipeptydil
aminopeptidase activity was evaluated with GlyPro-p-NA (Suárez Rendueles et al., 1981). The
Copyright 1999 John Wiley & Sons, Ltd.
1439
assay mixture was 0·33 m Gly-Pro-p-NA in
50 m Hepes–Tris, pH 7·0, containing 0·25% of
octyl-â-glucopyranoside. One unit of Ap or DPAP
activity corresponded to the amount of enzyme
that releases 1 ìmol/min of p-nitroanilide or
2-naphthylamine. Initial rates of substrate
hydrolysis releasing p-nitroanilide (å405 9620  1
cm 1) or 2-naphthylamine (å340 1700  1 cm 1)
were determined spectrophotometrically in a
Beckman DU 640 spectrophotometer. The activity
of samples eluted from the chromatography
columns was assayed by measuring the absorbance
of the reaction solution at fixed reaction times.
â-gal was determined with ONPG (Flores et al.,
1996), and invertase with sucrose (Flores et al.,
1994). One unit of â-gal or invertase activity
corresponded to the amount of enzyme that produces 1 ìmol of o-nitrophenol or glucose, respectively. All enzyme activities were determined at
37C. Protein was determined by the Lowry
method (Lowry et al., 1951).
Aminopeptidase activity in whole cells
For the determination of Ap activity in intact
and permeabilized whole cells, washed fresh cells
were suspended in 0·1  potassium phosphate
buffer at a cell concentration of 7·4108 cells/ml
and incubated for 15 min at 30C in the presence of
organic solvents or detergents. The suspension was
then centrifuged and the cells resuspended in phosphate buffer. Ap activity was determined by
mixing 0·4 ml cell suspension with 0·5 ml 100 m
Tris–HCl, pH 7·2. After 5 min, 0·1 ml of A–p–NA
stock solution was added and the reaction mixture
was incubated at 37C with shaking. After an
appropriate reaction time, the suspension was centrifuged and the absorbance of the supernatant
measured at 405 nm. Cell permeabilization was
assessed by measuring â-gal activity and the
fraction of methylene blue-stained cells (Flores
et al., 1994).
Preparation of spheroplasts
Washed fresh cells were suspended in 20 m
Tris–HCl, pH 7·8, containing 0·1  â-mercaptoethanol (5108 cells/ml) and incubated for
15 min at 30C. The suspension was centrifuged at
5000g for 15 min and cells resuspended in
20 m Tris–HCl, pH 7·2, containing 10 m MgCl2
and 0·6  KCl (similar results were obtained with
1·2  sorbitol instead of KCl). The digestion of
the cell wall was performed by adding lyticase
Yeast 15, 1437–1448 (1999)
1440
(120 U/ml) and incubating the cells at 30C with
gentle shaking (1·5% v/v glucuronidase can be used
instead). The progress of cell wall digestion was
followed by optical density after the dilution of
the cell suspension in water. After the incubation
period, the suspension was centrifuged at 3000g
for 10 min. The supernatant obtained was passed
through a PD-10 column equilibrated with buffer.
The eluate obtained was the periplasmatic fraction. The pelleted spheroplasts were resuspended
in 0·1  potassium phosphate buffer, pH 7·0,
briefly agitated and centrifuged at 5000g
for 10 min. The supernatant obtained was the
spheroplast lysate.
Biochemical techniques
Chromatographic procedures were performed
using a FPLC system (Pharmacia Biotech). Gel
filtration was performed at 5C in a Sephadex
G-200 column (0·945 cm, 30 ml gel) equilibrated and eluted with 20 m Tris–HCl, pH 7·2,
containing 0·2 m Co +2 and 0·02% sodium azide.
The flow rate was 0·125 ml/min and the fraction
volume 0·4 ml. Ion-exchange chromatography
was carried out at 5C with a DEAE Sepharose
CL-6B column (1·620 cm, 26 ml gel) equilibrated with 10 m Tris–HCl, pH 7·3, containing
0·2 m Co +2 and 0·02% sodium azide. After the
sample was applied, the column was washed with
80 ml of the same buffer and then eluted with a
linear gradient of NaCl (0–500 m in buffer). The
flow rate was 0·27 ml/min and fraction volume
0·8 ml.
Non-denaturing polyacrylamide gel electrophoresis was performed using a Mighty Small II
Unit (Hoefer SE 260), which was prepared
according to Nasir et al. (1981). Polymerization
of acrylamide was catalysed with riboflavin. A
voltage of 200 V was used and the electrophoresis
was carried out for 2 h at 20C. Aminopeptidase
activity staining was carried out by incubating
the gel for 30 min at room temperature with
Tris–HCl 5 m, pH 7·2 (0·5 m Co +2), containing 0·05% leucine-â-naphthylamide and 0·1% of
Fast Garnet. Isoelectric focusing was performed
with a pH gradient of 3·5–10 with a thin layer
of polyacrylamide gel, according to manufacturer
recommendations. Polymerization of acrylamide was catalysed with riboflavin. The staining procedure was similar to that described for
the non-denaturing polyacrylamide gel electrophoresis.
Copyright 1999 John Wiley & Sons, Ltd.
M. V. FLORES ET AL.
RESULTS
Endoproteinase activity
Results presented in Figure 1 show the existence
of proteolytic activity against acid-denatured
haemoglobin and Azocoll in cell extracts, both
of which increased during incubation at pH 5·0.
Azocoll activity was almost inhibited by PMSF,
while haemoglobin digestion was inhibited by
pepstatin (Table 1). These facts indicate the presence in K. lactis of endoproteinases activities similar to those described as PrA and PrB in S.
cereviseae. Activation results from the proteolytic
digestion of cytoplasmic specific inhibitors, which
bind firmly to the proteases upon cell disintegration (Lenney, 1975; Hirsh et al., 1989b). Activation patterns were similar for both enzymes. In
extracts of cells grown under C- or P-limitation,
initial activities were very low (i.e. no PrB activity
could be detected in fresh extracts from C-limited
cultures) and activation was completed after
about 50 h. In extracts from cells grown under
N-limitation, an appreciable initial activity was
measured which increased to a maximum level
within the first 20 h of incubation, after which a
fast inactivation of the enzyme activity occurred,
probably due to proteolytic digestion. The maximum PrA and PrB activities detected in N-limited
cultures were six-fold higher than those expressed
under C- or P-limitation (Table 1).
Carboxypeptidase activities
Carboxypeptidase yscY can be measured very
especifically with BTPNA (Aibara et al., 1971). As
shown in Figure 1, a similar activity could be
detected in fresh extracts, which also increased, by
incubation at pH 5·0. The effect of culture conditions on Cp expression was less pronounced in
comparison with PrA or PrB activities (Table 1).
The maximum Cp activity was measured in cells
grown in C-limited cultures. The extracts also
showed a high activity against N-Cbz-Phe-Leu, a
peptide-substrate that was hydrolysed at a high
rate by ysc CpY (Hayashi et al., 1973). As was the
case of the S. cerevisiae CpY, PMSF inhibits most
of the carboxypeptidase activity (measured either
with BTPNA or N-Cbz-Phe-Leu), while EDTA
had no effect on its activity. Biochemical analysis
of a S. cerevisiae CpY mutant had uncovered the
existence of an additional Cp activity, named CpS
(Wolf and Weiser, 1977; Wolf and Ehmann, 1981).
At variance with CpY, the activity of CpS,
Yeast 15, 1437–1448 (1999)
PROTEOLYTIC SYSTEM OF K. LACTIS
1441
measured with N-Cbz-Gly-Leu, was metaldependent and was inhibited by EDTA. We could
not detected carboxypeptidase activity against
N-Cbz-Gly-Leu in soluble extracts, either fresh or
activated, even in the presence of 2 m Zn +2.
Aminopeptidase activities
Ap activity was measured against Leu-p-NA
and Lys-p-NA because these substrates have been
commonly used to characterize Aps in other yeasts
(Frey and Röhm, 1978; Achstetter et al., 1982;
Hirsh et al., 1989a). The Ap activity can be
detected directly in the fresh extract. Furthermore,
no activation was achieved by incubation at
pH 5·0 (data not shown). Ap activity was higher
with Lys-p-NA than that with Leu-p-NA, increased two to threefold in presence of Co +2, and
was inhibited by Zn +2 and EDTA (Table 1). The
inhibitory effect of Zn +2 was less pronounced
when Leu-p-NA was the substrate. All extracts
showed the same ratio between Lys and Leu-p-NA
activities (about 2·0 in presence of Co +2), suggesting that similar Ap activities were expressed in
chemostat cultures under different nutrient limitations. Maximum Ap activities were found in
C-limited cultures. Soluble extracts also showed
a high activity towards Lys and Leu-ânaphthylamide, which increased in the presence
of Co +2.
Localization of the aminopeptidase activity
The ysc Ap II is the only protease that has been
reported to be partially localized in the periplasmatic space and appears to be involved in the
hydrolysis of exogenous peptides. This Ap showed
a strong activity towards Lys-p-NA (Frey and
Röhm, 1978; Hirsh et al., 1989a). To determine
whether an external activity was present in K.
lactis, the Ap activity was initially measured with
intact and permeabilized whole cells and compared
with the activity of a suspension of disrupted cells
(cell homogenate) that was taken as the maximum
value. Cell permeabilization was carried out with
different organic solvents and detergents and was
Figure 1. Activation of proteases in soluble extracts of K.
lactis by incubation at pH 5·0 and 25C. The soluble extracts (S
100 000 g) were prepared from yeast cells grown in chemostat at
D=0·1 h 1 under carbon (), nitrogen () or phosphate
limitation (). PrA, PrB and CpY activities were determined
with haemoglobin, Azocoll and N-benzoyl--tyrosine-pnitroanilide, respectively.
Copyright 1999 John Wiley & Sons, Ltd.
Yeast 15, 1437–1448 (1999)
1442
M. V. FLORES ET AL.
Table 1. Protease activities in soluble extracts of Kluyveromyces lactis NRRL 1118 grown
in a chemostat under different nutrient limitations.
Protease
Substrate
Ep (PrA)*
Haemoglobin
Ep (PrB)*
Azocoll
Cp*
N-Bz-L-Tyr-p-NA
N-Cbz-Phe-Leu
Ap**
N-Cbz-Gly-Leu
Leu-p-NA
Lys-p-NA
Leu-â-NA
Lys-â-NA
Activity
mU (mg protein) 1
Inhibitors/
activators
C-L
N-L
P-L
—
Pepstatin
—
PMSF
—
PMSF
—
PMSF
—
—
Co +2
Zn +2
EDTA
—
Co +2
Zn +2
EDTA
—
Co +2
—
Co +2
151
ND
0·6
ND
6·31
0·60
476
23
0
11·7
36·3
9·4
3·0
48·4
75·0
15·7
13·0
42·5
82·0
210
330
1533
120
11·5
<0·1
6·65
0·73
531
18
0
15·4
25·4
ND
ND
33
50
ND
ND
ND
ND
ND
ND
264
ND
1·67
ND
4·1
ND
ND
ND
ND
8·75
22
ND
ND
24
38
ND
ND
ND
ND
ND
ND
Proteolytic activities were determined in soluble extracts (S 100 000 g) obtained from cells grown at
D=0·1 h 1 under carbon (C-L), nitrogen (N-L) or phosphate (P-L) limitation.
Ep, endoproteinase; Cp, carboxypeptidase; Ap, aminopeptidase.
*Maximum specific activity in soluble extracts activated at pH 5·0. **Activity in fresh extracts (Ap was
not activated by incubation at pH 5·0). Before the activity assay, the soluble extracts were incubated
for 5 min in the presence of 0·5 m Co +2, Zn +2, or for 30 min in the presence of 5 m EDTA, 2 m
PMSF or 0·1 m pepstatin. Cations and inhibitors were also present in the reaction mixture. For
calculation of specific activities, the protein values of the fresh extracts before acidification were taken.
The protein concentrations were 15·1, 7·11 and 11·55 mg/ml for C-L, N-L and P-L extracts,
respectively. Loss of protein after acidification of the fresh extract at pH 5·0 and centrifugation was
20–30%. ND, not determined.
assessed by measuring the fraction of methylene
blue-stained cells and the â-gal activity. Results
obtained showed that, with intact cells, 40% of
the Leu-p-NA activity of homogenate could be
measured. By contrast, Lys-p-NA was hydrolysed
at a very low rate (3% relative activity) (Table
2). An attempt was made to measure the Ap
activity in permeabilized cells. Although the Ap
activity was enhanced when cells were treated with
toluene, Triton X-100 or chloroform (in comparison with the activity of intact cells), maximum
Ap activities could not be achieved using permeabilized cells, indicating that the Ap activity
was partially inactivated by the solvents and
Copyright 1999 John Wiley & Sons, Ltd.
surfactants tested. In the case of Triton X-100,
the low Ap activity could be associated with an
insufficient permeabilization, because the activity
correlated with the fraction of stained cells.
Optimization of permeabilization with Triton
X-100, in connection with the Ap activity,
warrants further research.
To determine whether the activity displayed
by intact cells was actually due to periplasmatic
activity, cells were converted to spheroplasts by
using cell wall-lytic enzymes. â-gal and invertase
were used as marker enzymes for the intracellular
and the periplasmatic localizations, respectively.
Results depicted in Table 3 show that the Ap
Yeast 15, 1437–1448 (1999)
1443
PROTEOLYTIC SYSTEM OF K. LACTIS
Table 2. Aminopeptidase activity in intact and permeabilized whole cells of Kluyveromyces lactis NRRL 1118.
Stained
cellsa
(%)
Intact cells
Permeabilized cells
NLS 0·2%
CTAB 0·2%
Triton X-100 0·2%
Toluene 2%
Chloroform 2%
Cell homogenateb
0
100
100
42·4
100
100
—
Enzyme activity [mU (mg cell protein) 1]
Ap
Leu-p-NA
Ap
Lys-p-NA
<0·1
2·32
0·718
14·55
1·97
6·4
15·2
16·46
15·1
0·96
0·12
3·35
4·24
1·935
5·83
â-gal
10 3
0·64
<0·1
6·49
9·22
4·86
21·8
Ap was determined in cells growing in carbon-limited chemostat cultures at D=0·1 h 1. Biomass from
the culture media was recovered by centrifugation at 5C (5000g for 10 min), washed twice with
0·1  potassium phosphate buffer, pH 7·0, and resuspended in buffer at a cell concentration of
7·4108 cells/ml (3·7 mg dry biomass/ml, biomass protein, 44%). Permeabilization was carried out
by incubating the cell suspensions with solvents or detergents in the concentration indicated for 10 min
at 30C with shaking. Cells were then centrifuged, washed twice and resuspended to original volume
with buffer. Ap activity was determined by mixing 0·4 ml of the untreated or permeabilized cell
suspension with 0·68 ml Tris–HCl 100 m, pH 7·2, and 0·12 ml of A-p-NA (stock solution). After
20 min of incubation at 37C, cells were centrifuged in a microfuge (14 000g for 1 min) and the
absorbance of the supernatant measured at 405 nm.
a
Methylene blue test. bCell suspension disrupted with glass beads.
NLS, N-lauroylsarcosine. CTAB, cetyltrimethylammonium bromide (Cetrimide).
Table 3. Distribution of enzyme activities in cellular fractions of Kluyveromyces lactis
NRRL 1118.
Cellular fraction
Periplasmatic*
Spheroplast lysate
Spheroplast pellet
Cell homogenate**
S 20 000g
S 100 000g
P 100 000g
Protein Ap Lys-p-NA Ap Leu-p-NA DPAP Invertase â-gal
(mg/ml)
(mU/ml)
(mU/ml)
(mU/ml) (U/ml) (U/ml)
ND
ND
ND
24·0
18·5
11·5
ND
Neg
25
1·1
543
595
552
18
Neg
7·5
Neg
140
165
141
5·0
ND
ND
ND
ND
26·5
15·5
7·2
7·9
0·36
0·28
180
225
191
ND
0·67
15·3
0·94
370
342
400
2·5
The spheroplasts and cell homogenates were prepared from cells growing in the carbon-limited
chemostat at D=0·1 h 1, as described in Materials and Methods.
*Activity released during spheroplast formation (5108 cells/ml).
**Cell suspension disrupted with glass beads (1010 cells/ml). S, supernatant; P, pellet; Ap,
aminopeptidase activity; DPAP, X-prolyl-dipeptidyl-aminopeptidase activity (Gly-Pro-p-NA); Neg,
negligible; ND, not determined.
activity (measured with either Leu or Lys-p-NA)
was almost recovered in the spheroplast lysate. We
conclude that the Ap activity is located intracellularly, and that the high activity of intact cells with
Leu-p-NA and the minor activity with Lys-p-NA
Copyright 1999 John Wiley & Sons, Ltd.
was due to the free diffusion of these substrates
to the interior of the cell. The differential centrifugation of a crude extract also showed that
the Ap activity remained in the soluble fraction
(Table 3).
Yeast 15, 1437–1448 (1999)
1444
M. V. FLORES ET AL.
In addition to Aps, dipeptidyl aminopeptidases
(DPAPs) have been well-characterized in S.
cerevisiae. They are highly active against substrates having the structure X-prolyl-p-nitroanilide
(typically Ala-Pro and Gly-Pro-p-NA) and were
found to be associated with the vacuolar membrane (DPAP-B) and the plasmatic membrane
(DPAP-A) (Garcı́a Alvarez et al., 1985; Bordallo
et al., 1990; Roberts et al., 1992). The presence of
this activity was detected in fresh extracts (under
all culture conditions), using the dipeptide GlyPro-p-NA (Table 3). The DPAP activity was not
enhanced by Co +2. Furthermore, differential centrifugation of a cell-free extract showed that
50% of the X-prolyl-dipeptidyl aminopeptidase
activity is associated with a particulate fraction
(P 100 000g). These results are in agreement
with those described in S. cerevisiae (Suárez
Rendueles et al., 1981).
Partial characterization of the aminopeptidase
activity
Aps in fresh extracts were partially characterized
by gel exclusion, ion-exchange chromatography
and non-denaturing electrophoretic methods.
When the fractions eluted from the G-200 column
were analysed with either Leu-p-NA or Lys-p-NA,
only one peak of activity was found. The estimated
molecular weight was 60 000 Da (Figure 2). On
the other hand, ion-exchange chromatography
separated the Ap activity into two peaks (no
activity was found in the unbound fraction)
eluting at 133 and 213 mM of NaCl, respectively
(Figure 3). Substrate specificity of these two peaks
against different A-p-NAs is shown in Table 4. The
typical substrate for DPAP, Gly-Pro-p-NA, and
substrates in which N-terminal was blocked, were
not hydrolysed.
The Ap activity in crude extracts was also characterized by non-denaturing gel electrophoresis
and isoelectric focusing, which were stained for
activity on leu and lys-â-naphthylamide. Only one
activity band was visible on denaturing gel electrophoresis (Figure 4 shows the band active only
against leu-â-NA). However, isoelectric focusing
demonstrated the presence of two bands which
focused at close pI values (pI 4·82 and 5·1) (Figure
5 shows bands active only against leu-â-NA).
DISCUSSION
Our findings demonstrate that K. lactis expressed
proteolytic activities similar to those found in S.
Copyright 1999 John Wiley & Sons, Ltd.
Figure 2. Separation of K. lactis aminopeptidases by gel
filtration chromatography on Sephadex G-200. Sample: 1 ml of
a soluble extract (S 100 000 g) prepared from cells grown in
carbon-limited chemostat at D=0·1 h 1, Ap activity 690 mU/
ml (0·6 m Lys-p-NA, 0·5 m Co +2). Gel filtration was done at
5C in a column (0·945 cm, 30 ml gel) equilibrated and eluted
with 20 m Tris–HCl, pH 7·2, containing 0·2 m Co +2 and
0·02% sodium azide. The flow rate was 0·125 ml/min and the
volume fraction 0·4 ml. Ap activity of collected fractions was
determined with 0·6 m leu or lys-p-NA in 50 mM Tris–HCl,
pH 7·2, 0·5 m Co +2. Molecular weight markers: Apo, apoferritin (443 kDa); Cat, catalase (232 kDa); Ald, aldolase
(158 kDa); Alb, albumin (66 kDa), Rib, ribonuclease
(13·7 kDa). , lys-p-NA (mU/ml); , leu-p-NA (mU/ml).
cerevisiae. This fact was particularly evident in the
case of proteases such as PrA, PrB and CpY with
regard to substrate specificity, activation at pH 5·0
and inhibition patterns. Other well characterized
Yeast 15, 1437–1448 (1999)
1445
PROTEOLYTIC SYSTEM OF K. LACTIS
Table 4. Characteristics of aminopeptidase fractions
eluted from ion-exchange chromatography on Sepharose CL-6B
Relative Ap activity
Substrates
Peak 1
Peak 2
Co +2
EDTA
PMSF+Co +2
Zn +2
—
-leucine-p-NA
Co +2
EDTA
PMSF+Co +2
Zn +2
—
-methionine-p-NA Co +2
-alanine-p-NA
Co +2
-arginine-p-NA
Co +2
-glycine-p-NA
Co +2
-ã-glutamyl-p-NA Co +2
-proline-p-NA
Co +2
N-Cbz-Arg-p-NA Co +2
Gly-Pro-p-NA
Co +2
100*
0
100
11
77
92
0
92
17
60
63
49
40
2·5
2·5
0·64
0
0
100*
12
100
4
79
27
ND
27
31·6
ND
53
43
28
1·1
ND
0·83
0
0
-lysine-p-NA
Figure 3. Separation of K. lactis aminopeptidases by ionexchange chromatography on DEAE Sepharose CL-6B.
Sample: 2 ml of a soluble extract (S 100 000 g) prepared from
cells grown in carbon-limited chemostat at D=0·1 h 1, Ap
activity 690 mU/ml (0·6 m Lys-p-NA, 0·5 m Co +2). Ionexchange chromatography was carried out at 5C in a column
(1·620 cm, 26 ml gel) equilibrated with 10 m Tris–HCl,
pH 7·3, containing 0·2 m Co +2 and 0·02% sodium azide. After
the sample was applied, the column was washed with 80 ml of
the same buffer and then eluted with a linear gradient of NaCl
(0–500 m in buffer). The flow rate was 0·27 ml/min and the
volume fraction 0·8 ml. Ap activity of collected fractions was
determined with 0·6 m leu or lys-p-NA in 50 m Tris–HCl,
pH 7·2, 0·5 m Co +2. , lys-p-NA (mU/ml); , leu-p-NA
(mU/ml); —— gradient of NaCl.
carboxypeptidase activity in S. cerevisiae is the
vacuolar CpS, a metal-dependent protease (Wolf
and Weiser, 1977; Wolf and Ehmann, 1981;
Spormann et al., 1991). We could not detect the
presence of a CpS activity in either fresh or activated cell-free extracts, using the dipeptide N-CbzGly-Leu, even in presence of Zn +2. Further
research is needed to establish whether this activity
is absent in K. lactis or could be detected under
other growth conditions.
A multiplicity of Aps have been detected by
biochemical methods in cell-free extracts of different S. cerevisiae strains, and some of them have
been confirmed by genetic studies using appropriate mutants. In this yeast, at least three Ap activities were separated by gel filtration on Sepharose
6B (or Sephadex G-200) or by ion-exchange
chromatography on Sepharose CL-6B, when the
Copyright 1999 John Wiley & Sons, Ltd.
Separation of aminopeptidase activities was done on DEAESepharose CL-6B, as described in Figure 3. Before the activity
assay, the samples were incubated for 5 min in the presence of
0·5 m Co +2 or Zn +2 or for 30 min in the presence of 5 m
EDTA or 2 m PMSF. Cations and inhibitors were also
present in the reaction mixture. In all cases, the reaction
mixture contained 0·01 m Co +2 derived from the elution
buffer.
*Activities of peak 1 and peak 2 with Lys-p-NA in presence of
0·5 m Co +2 were 70·2 and 105 mU/ml, respectively. Values in
the table are related to these activities. ND, not determined.
eluted fractions were assayed with Leu and Lysp-NA (Frey and Röhm, 1978; Achstetter et al.,
1983; Trumbly and Bradley, 1983). Nondenaturing polyacrylamide gel electrophoresis, followed by Ap activity staining, also revealed two
major Lys-â-NA splitting activities which also
react with Leu-â-NA, and one additional slowly
migrating activity band that appeared only after
incubation with Leu-â-NA (Trumbly and Bradley,
1983; Hirsh et al., 1989a). Among these Ap activities detected by biochemical methods, the best
characterized is ApI, a vacuolar Ap with a molecular mass of 640 kDa. ApI is typical leucineaminopeptidase, which is highly active towards
Leu-p-NA but reacts weakly towards Lys-p-NA,
and is activated by Zn +2. The other Ap activities
corresponded to proteins with molecular masses in
Yeast 15, 1437–1448 (1999)
1446
Figure 4. Activity staining of aminopeptidase activities from
K. lactis on non-denaturing polyacrylamide gel electrophoresis.
Non-denaturing polyacrylamide gel electrophoresis was performed as described by Nasir et al. (1981). Polymerization of
acrylamide was catalysed with riboflavin. A voltage of 200 V
was used and the electrophoresis was carried out for 2 h at
20C. Aminopeptidase activity staining was carried out by
incubating the gel for 30 min at room temperature with Tris–
HCl 5 m, pH 7·2, 0·5 m Co +2 containing 0·05% leucine
â-naphthylamide and 0·1% of Fast Garnet. (A) 50 ìl of soluble
extract (S 100 000 g) prepared from cells grown in carbonlimited chemostat at D=0·1 h 1, Ap activity 650 mU/ml
(0·6 m Lys-p-NA, 0·5 m Co +2). (B) 50 ìl peak eluted from
G-200, Ap activity 100 mU/ml (0·6 m Lys-p-NA, 0·5 m
Co +2).
the range 40–100 kDa, which are active against
Leu and Lys-p-NA. ApII preferentially hydrolysed
Lys-p-NA and is partly periplasmatic. The other
known Ap is ApCo, which is active only in the
presence of Co +2 and preferentially hydrolyses
Lys-p-NA (Achstetter et al., 1982). This enzyme
has not been purified yet, but a recently purified
new Ap, namely ApY (molecular mass 70 kDa),
located in the vacuole, appears to be the precursor
of ApCo (Yasuhara et al., 1994). Hydrolysis of
amino acid-4-methylcoumaryl-7-amides by ApY
was markedly enhanced by Co +2. In S. pombe,
only one Ap activity was detected in cell-free
extracts by gel filtration chromatography that
showed the highest activity against Lys-p-NA
(Suárez Rendueles et al., 1991). We used similar
biochemical techniques to characterized Ap activities in cell-free extracts of K. lactis. Although we
were unable to give the exact number of Aps
present in the yeast cell, our results show that K.
Copyright 1999 John Wiley & Sons, Ltd.
M. V. FLORES ET AL.
Figure 5. Activity staining of aminopeptidase activities from
K. lactis on isoelectric focusing (IEF), which was performed
with a pH gradient of 3·5–10 with a thin layer of polyacrylamide gel, according to the manufacturer’s recommendations.
Polymerization of acrylamide was catalysed with riboflavin.
Aminopeptidase activity staining was carried out by incubating the gel for 30 min at room temperature with Tris–HCl
5 m, pH 7·2, 0·5 m Co +2, containing 0·05% leucine
â-naphthylamide and 0·1% of Fast Garnet. (A) IEF markers, pI
isoelectric points, Coomasie blue staining. (B) 50 ìl of soluble
extract (S 100 000 g) prepared from cells grown in carbonlimited chemostat at D=0·1 h 1, Ap activity 650 mU/ml
(0·6 m Lys-p-NA, Co +2 0·5 m).
lactis contains at least two major intracellular Ap
activities that are different from those reported in
other yeasts. Both activities are enhanced by Co +2
and inhibited by EDTA. Among different
aminoacyl-p-NAs, they preferentially hydrolyse
Lys-p-NA. The molecular mass of this activities
were similar, about 60 kDa and showed a similar
behaviour in non-denaturing polyacrylamide electrophoresis. In addition, isoelectric focusing of cell
extracts also revealed the presence of two main Ap
activities located at close pI values. These findings
suggest that Ap activities are carried out by closely
related proteins.
Taking advantage of the use of chemostat cultures, which offer the advantage of well-defined
growth conditions, we demonstrated that PrA and
PrB were expressed at high levels when cells were
grown under N-limitation, in comparison with
those activities expressed in cells grown under
carbon or phosphate limitation. The effect of
growth conditions was much less pronounced on
Yeast 15, 1437–1448 (1999)
PROTEOLYTIC SYSTEM OF K. LACTIS
the expression of Cp and Ap activities. The level of
Aps was even slightly higher in C-limited cultures.
The primary role of vacuolar proteases seems to be
the degradation of the bulk of cell protein to yield
amino acids for protein synthesis. This function is
more critical under nutritional stress to facilitate
cellular restructuring, i.e. during sporulation, or
for survival under conditions of nutrient starvation
(Teichert et al., 1989; Hilt and Wolf, 1992; Bart
Van Den Hazel et al., 1996; Lee and Goldberg,
1996). PrA seems to play a special role under these
conditions, demonstrated by the fact that lack of
this enzyme leads to cell death upon nitrogen
starvation of cells. The central function of PrA
cannot be substituted by PrB. In starvation experiments, cells growing in the exponential growth
phase of a batch culture are separated from the
growth medium and resuspended in medium lacking the component for which starvation behaviour
is to be measured. Under such conditions, growth
is practically absent and cellular functions are only
necessary for the maintenance of cell viability. In
chemostat cultures, vegetative growth is maintained at a steady state, but restricted by some
nutrient, which is supplied in limiting amounts.
Thus, cell physiology is expected to be different
under the conditions used in starvation experiments and those found in chemostat cultures.
Nevertheless, our findings suggest that PrA and
PrB activities also play a key physiological role
when vegetative growth is restricted, in this case
by nitrogen limitation. Considering that the function of Cp and Ap activities is to degrade the
peptides, originated by PrA and PrB activities,
into the respective amino acids (Teichert et al.,
1989; Bart Van Den Hazel et al., 1996), the
fact that levels of these enzymes are not induced
by N-limitation suggests that hydrolysis of protein
to peptides might be the limiting step in the
pathway of general protein degradation in the
vacuole.
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