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Journal of Bacteriology, December 1998, p. 6736-6742, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Disruption of PMR1, Encoding a
Ca2+-ATPase Homolog in Yarrowia lipolytica,
Affects Secretion and Processing of Homologous and Heterologous
Proteins
Young-Sun
Sohn,1,2
Cheon Seok
Park,3
Sun-Bok
Lee,4 and
Dewey D. Y.
Ryu1,2,*
Biochemical Engineering Program, Department
of Chemical Engineering and Material Science1
and
Microbiology Graduate Group,2
University of California, Davis, California 95616;
School
of Pharmacy, University of Wisconsin, Madison, Wisconsin
537063; and
Pohang Institute of
Science and Technology, Pohang, Korea4
Received 11 May 1998/Accepted 1 October 1998
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ABSTRACT |
The Yarrowia lipolytica PMR1 gene (YlPMR1)
is a Saccharomyces cerevisiae PMR1 homolog which encodes a
putative secretory pathway Ca2+-ATPase. In this study, we
investigated the effects of a YlPMR1 disruption on the
processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The
secretion of 32-kDa mature alkaline extracellular protease (AEP) was
dramatically decreased, and incompletely processed precursors were
observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa
premature AEP were secreted, and an intracellular 52-kDa premature AEP
was also detected. The acid extracellular protease activity of the
YlPMR1-disrupted mutant was increased by 60% compared to
that of the wild-type strain. The inhibitory effect of mutations in
secretory pathway Ca2+-ATPase genes on the secretion of
rice 
-amylase was also observed in the Y. lipolytica and
S. cerevisiae PMR1-disrupted mutants. Unlike rice
-amylase, the secretion of Trichoderma reesei
endoglucanase I (EGI) was not influenced by the YlPMR1
disruption. However, the secreted EGI from the
YlPMR1-disrupted mutant had different characteristics than
that of the control. While wild-type cells secreted the
hyperglycosylated form of EGI, hyperglycosylation was completely absent
in the YlPMR1-disrupted mutant. Our results indicate that
the effects of the YlPMR1 disruption as manifested by the
phenotypic response depend on the characteristics of the reporter
protein in the recombinant yeast strain evaluated.
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INTRODUCTION |
In living cells, the control of
intracellular calcium (Ca2+) concentrations is critically
important to the regulation of cellular processes such as muscle
contraction, neurotransmitter release, and cell proliferation
(1). In addition, Ca2+ is involved in the
transport of secretory proteins from the endoplasmic reticulum (ER)
(30). Intracellular compartments involved in the secretory
pathway, particularly the ER and Golgi apparatus, contain higher
concentrations of Ca2+ (~1 mM) than the cytoplasm (
0.1
µM) under normal conditions (1, 28). This Ca2+
concentration differential is normally maintained by
Ca2+-ATPases, and a massive Ca2+ efflux from
subcellular compartments to the cytoplasm can cause a specific cellular
response depending on the external signal.
Two types of Ca2+-ATPases, plasma membrane
Ca2+-ATPases (PMCA) and sarco/endoplasmic
reticulum Ca2+-ATPases (SERCA), have been classified
based on genetic and biochemical analyses (7). Recently, the
presence of a new type of Ca2+-ATPase distinct from PMCA
and SERCA has been proposed (11). Rudolph et al. identified
a novel P-type ATPase encoded by PMR1 which functions as a
Ca2+ pump and affects transit through the secretory pathway
in the yeast Saccharomyces cerevisiae (29). The
PMR1 gene is localized to the Golgi apparatus and was shown
to be required for normal Golgi function (2). Sorin et al.
provided biochemical evidence that Pmr1 is indeed a Golgi-specific
Ca2+ pump and is distinct from both SERCA and PMCA
(31). Interestingly, the PMR1-disrupted mutant
displays pleiotropic changes, such as a Ca2+-dependent
growth defect, secretion of an unprocessed factor, suppression of
various sec mutants blocked in ER and/or Golgi and
post-Golgi transport, and incomplete outer-chain glycosylation (2,
13). These phenotypes can be reversed by the addition of a high
concentration of Ca2+ (10 mM) to the medium, implicating a
direct role for exogenous calcium in Golgi function (2).
Furthermore, disruption of the PMR1 gene resulted in a 5- to
50-fold increase in the secretion of bovine prochymosin, a bovine
growth hormone, and a nonglycosylated variant of human urinary
plasminogen activator (14, 29). In contrast, secretion of
the plant protein thaumatin could not be improved to any significant
extent by disruption of the PMR1 gene (14). These
results indicate that the PMR1 gene product plays an
important role in the yeast secretory pathway.
Yarrowia lipolytica secretes high levels of several
extracellular enzymes, including alkaline extracellular protease (AEP), RNase, lipase, and acid proteases (5, 15). In fact, AEP is secreted at a level of more than 1 g/liter under optimal conditions. The high-level secretion capacity of Y. lipolytica coupled
with detailed studies on AEP secretion and processing make this yeast strain an excellent model system for studying protein secretion (22, 23).
Recently, we have cloned the Y. lipolytica PMR1 gene
(YlPMR1), which is a Saccharomyces cerevisiae
PMR1 homolog, and characterized the YlPMR1-disrupted
mutant in the yeast Y. lipolytica (26). In the
present paper, the effects of the YlPMR1 disruption on the
processing and secretion of homologous and heterologous proteins are
described. AEP, acid extracellular protease (AXP), rice -amylase, and Trichoderma reesei endoglucanase I (EGI) were used as
reporter proteins to illustrate the findings.
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MATERIALS AND METHODS |
Strains and media.
The yeast strains and plasmids used in
this work are described in Table 1. The
SMS397A (MATa ade1 ura3 xpr2) strain derived
from Y. lipolytica CX161-1B (MATa
ade1; ATCC 32338) was used to construct the
YlPMR1-disrupted mutant designated CS3 (26). The
Escherichia coli strain DH5 was used for plasmid DNA
propagation and subcloning (12). The S. cerevisiae
PMR1-disrupted mutant (AA274) and its isogenic wild-type strain
(AA255) were kindly provided by G. R. Fink (Massachusetts
Institute of Technology) (2). Y. lipolytica and
S. cerevisiae cultures were maintained on YM medium (0.3%
Bacto yeast extract, 0.3% Bacto malt extract, 0.5% Bacto Peptone, 1%
dextrose, 2% agar) and cultivated in YPD medium (yeast extract, 10 g/liter; Bacto Peptone, 10 g/liter; glucose, 20 g/liter). The
production medium used for recombinant -amylase and EGI was GPP (10 g of glycerol/liter, 3.4 g of yeast nitrogen base/liter without
amino acids and ammonium sulfate, 3.4 g of Proteose Peptone/liter,
50 mg of uracil/liter, 50 mg of adenine/liter in 50 mM sodium phosphate
buffer [pH 6.8]).
DNA manipulation and transformation.
General recombinant DNA
techniques were performed as described in Sambrook et al.
(30). E. coli transformation was performed by the
SEM method (16). S. cerevisiae and Y. lipolytica transformations were carried out by the lithium acetate
method as described by Ito et al. (17) and Gaillardin et al.
(9), respectively, with HaeIII-digested E. coli DNA as the carrier DNA.
Construction of expression vectors.
Construction of the
vectors pXOS103-In and pXCSIn(myc) for expression in Y. lipolytica of rice -amylase and fungal EGI, respectively, was
done as described by Park et al. (25, 27). The rice
-amylase expression vector for S. cerevisiae, pSCRA, was
constructed by inserting the rice -amylase coding sequence
(HindIII fragment of pENO103 [19]) into
pDB20, a 2µm vector containing the URA3 gene as a
selection marker (4). The endoglucanase expression vector,
pSCFC, was constructed by transferring the EGI coding sequence
(HindIII fragment) from pXCS to pDB20.
SDS-PAGE and Western blot analysis.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted as
described by Laemmli (20). After separation by SDS-PAGE,
proteins were electroblotted onto a nitrocellulose membrane (Schleicher
& Schuell, Inc., Keene, N.H.) in ice-cold transferring buffer (15.6 mM
Tris, 120 mM glycine, 20% methanol [pH 8.3]) at 100 V for 1 h.
The primary antibodies for Western blot analysis were anti-AEP,
provided by D. M. Ogrydziak, anti-AXP provided by T. W. Young
(University of Birmingham, Birmingham, United Kingdom), anti c-myc
antibody (Invitrogen, San Diego, Calif.), and anti-barley -amylase
antibody provided by S. Katoh and M. Terashima (Kyoto University,
Japan). The secondary antibodies were anti-mouse immunoglobulin G (IgG)
and anti-rabbit IgG antibodies conjugated with peroxidase as supplied
in the Phototope-HRP Western Blot Detection Kit (New England Biolabs).
The detection procedure was performed according to the manufacturer's
recommendation. The Western blot analyses were performed for AEP from
SMS397A and CS3 cells grown in GPP medium at pH 6.8 and 5.0, respectively, AEP from SMS397A and CS3 cells grown in YPD medium
supplemented with 10 mM CaCl2, AXP from SMS397A and CS3
cells grown in GPP medium at pH 6.8 and 5.0, respectively, and rice
-amylase from SMS397A-RA and CS3-RA grown in YPD medium supplemented
with 10 mM CaCl2. The protein samples were prepared by
concentrating the culture supernatant and cell extracts 10-fold with
trichloroacetic acid precipitation before loading. Ten microliters of
the concentrated culture supernatants and cell extracts containing
approximately 0.15 µg of total protein was loaded.
Preparation of cell extracts.
After the cells were grown in
GPP or YPD medium for 36 h, 5 ml of cell suspension was collected
and centrifuged. Cell pellets were dissolved with 0.5 ml of ice-cold
protease inhibitor cocktail solution (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.) and disrupted by vortexing with 1 g of
acid-washed glass beads (diameter, 212 to 300 µm; Sigma Chemical Co.,
St. Louis, Mo.). Ten microliters of crude extracts were mixed with 2.5 µl of 5× sample loading buffer, boiled for 5 min, and loaded onto an
SDS-PAGE gel.
Enzyme activity assays.
The acid protease activity was
estimated by measuring the hydrolysis of the standard hemoglobin by the
method described by Larson and Whitaker (21). The substrate
contained acid-denatured bovine hemoglobin (Sigma Chemical Co.) at a
final concentration of 5 g/liter in 0.05 M acetate-0.05 M
phosphate-5 × 10
6 M EDTA buffer adjusted to pH
3.5. Enzyme solution (0.4 ml) and substrate (3.0 ml) were combined and
incubated for 1 h at 25°C. After the reaction was stopped with
3.0 ml of 10% trichloroacetic acid, the precipitate was filtered and
1.0 ml of the filtrate was assayed by using the Bio-Rad Protein Assay
Kit (Bio-Rad Laboratories). One enzyme unit corresponds to the amount
of protease causing an increase in absorbance of 0.1 at 595 nm after
1 h.
The starch-degrading activity of recombinant rice -amylase was
determined by monitoring reducing sugars by the modified
dinitrosalicylic acid (DNS) method (27). Enzyme solution
(0.5 ml) was added to 0.5 ml of substrate solution (100 mM sodium
acetate buffer with 5 mM CaCl2 and 1% soluble starch [pH
5]). After 10 min of incubation at 30°C, the reaction was terminated
by adding 0.5 ml of DNS to 0.5 ml of reaction solution and boiling for
5 min. The solution was then diluted with 4 ml of distilled water, and
absorbance was measured at 540 nm.
The carboxymethyl cellulose activity of recombinant EGI was determined
by monitoring reducing sugars by the DNS method (
3,
24). The
reaction was initiated by adding 0.2 ml of enzyme solution
to 1.8 ml of
substrate (1% carboxymethyl cellulose in 50 mM sodium
citrate [pH
4.8]). After incubation for 20 min at 50°C, 3 ml of
DNS solution was
added to terminate the reaction. Following 5
min of boiling, the
absorbance at 540 nm was
determined.
For both of these assays, glucose solution was used as a standard. One
enzyme unit corresponds to the amount of enzyme required
to produce 1 µmol of reducing sugar from the substrate per
min.
Protein assays.
The Bio-Rad Protein Assay Kit was used for
protein assays with bovine serum albumin as the standard. Absorbance at
595 nm was used to monitor the protein content of culture supernatants and cell extracts.
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RESULTS |
AEP processing and secretion in a Ylpmr1-disrupted
mutant.
To examine the functional role of the YlPMR1
gene product with secretory proteins in Y. lipolytica, the
effects of the YlPMR1 disruption on the secretion of
endogenous proteins were investigated. SDS-PAGE results showed that the
productivity of 32-kDa mature AEP, a major secreted protein in Y. lipolytica, was reduced dramatically in the CS3 strain compared to
that in the wild-type SMS397A strain (data not shown).
Western blot analysis with anti-AEP antibody showed that the processing
and secretion of AEP in a CS3 strain grown in GPP
medium was
significantly affected by the
YlPMR1 disruption (Fig.
1, cf. the 32-kDa mature AEP bands in
lanes 1 and 2 with those
in lanes 3 and 4 at pH 6.8; also compare lanes
5 and 6 with lanes
7 and 8 at pH 5.0). The CS3 strain secreted 52- and
36-kDa AEP
precursors in addition to the 32-kDa mature AEP (Fig.
1,
lane
3), while the SMS397A strain secreted a large amount of only the
32-kDa mature AEP at pH 6.8 (Fig.
1, lane 1). Matoba et al. suggested
that the 52- and 36-kDa polypeptides were possible precursors
for the
32-kDa mature AEP (
22). The 32-kDa mature AEP proteins
in
the samples taken from both the supernatant and cell extract
of the CS3
strain almost completely disappeared at pH 5.0 (Fig.
1, lanes 7 and 8),
while significant amounts of secreted and intracellular
mature AEP were
detected in the SMS397A strain at pH 5.0 (Fig.
1, lanes 5 and 6). The
intracellular 52-kDa AEP precursor was
found in the sample taken from
the cell extract of the CS3 strain
cultivated at pH 6.8 (Fig.
1, lane
4), while it was not detected
in the cell extract sample from the
wild-type SMS397A strain (Fig.
1, lane 2).
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FIG. 1.
Western blot analysis of AEP from SMS397A
(YlPMR1) and CS3 (Ylpmr1) strains grown in GPP
medium at pH 6.8 and 5.0, respectively. Lanes 1 and 5, supernatants of
SMS397A culture grown at pH 6.8 and 5.0, respectively; lanes 2 and 6, cell extracts of SMS397A culture grown at pH 6.8 and 5.0, respectively;
lanes 3 and 7, supernatants of CS3 culture grown at pH 6.8 and 5.0, respectively; lanes 4 and 8, cell extracts of CS3 culture grown at pH
6.8 and 5.0, respectively. mAEP, mature AEP; pAEP, AEP precursors. The
test for secreted proteins is shown in lanes 1, 3, 5, and 7, and the
test for the intracellular proteins is shown in lanes 2, 4, 6, and 8.
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A very significant effect of pH on the secretion of the 32-kDa AEP and
on the intracellular processing of the 36-kDa precursor
AEP in the CS3
strain was also found. The amount of the 32-kDa
mature AEP secreted by
SMS397A decreased significantly when the
pH was lowered from 6.8 to 5.0 (Fig.
1, cf. lanes 5 and 1), while
the amount of the intracellular
32-kDa mature AEP increased (Fig.
1, cf. lanes 6 and 2). The secreted
32-kDa mature AEP and the
36- and 52-kDa precursor AEPs from the CS3
strain disappeared
when the pH was lowered from 6.8 to 5.0 (Fig.
1, cf.
lanes 7 and
3). Almost all of the intracellular 32-kDa mature and
36-kDa precursor
AEP disappeared when the pH was lowered from 6.8 to
5.0 (Fig.
1, cf. lanes 8 and
4).
The secretion of the 32-kDa mature AEP in the CS3 strain was restored
when the YPD medium was supplemented with 10 mM CaCl
2 (Fig.
2). Both the extracellular and
intracellular 32-kDa mature
AEPs were not detected in the CS3 strain at
a low-calcium concentration
compared to the detection of these
proteases in the SMS397A strain
(Fig.
2, cf. lanes 3 and 4 with lanes 1 and 2). When 10 mM CaCl
2 was added to the YPD medium,
secretion of the 32-kDa mature AEP
and the 36-kDa premature AEP was
restored in the CS3 strain (Fig.
2, cf. lanes 7 and 3), while the
intracellular 32-kDa mature AEP
and the 36-kDa premature AEP were not
detectable (Fig.
2, cf.
lanes 8 and 4) regardless of the concentration
of calcium added
to the medium. The amount of the 32-kDa intracellular
mature AEP
significantly decreased in the SMS397A strain at the higher
calcium
concentration (Fig.
2, cf. lanes 6 and 2).
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FIG. 2.
Western blot analysis of AEP from SMS397A
(YlPMR1) and CS3 (Ylpmr1) strains grown in YPD
medium supplemented with 10 mM CaCl2 at pH 6.8. The Western
blot experiment was performed with anti-AEP antibody. Lanes 1 and 5, supernatants of SMS397A culture grown in low-calcium (~180 µM) and
high-calcium (10 mM) concentration media, respectively; lanes 2 and 6, cell extracts of SMS397A culture grown in low- and high-calcium
concentration media, respectively; lanes 3 and 7, supernatants of CS3
culture grown in low- and high-calcium concentration media,
respectively; lanes 4 and 8, cell extracts of CS3 culture grown in low-
and high-calcium concentration media, respectively.
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These results suggest that the
YlPMR1 disruption in
Y. lipolytica significantly alters the pH and Ca
2+
effects on the processing and secretion patterns of mature and
premature AEP
proteins.
Activation of secreted AXP.
The effects of the
YlPMR1 disruption in the CS3 strain on the secretion and
processing of AXP were also studied. The AXP activity (92.0 U/ml) of
the CS3 strain was about 60% higher compared to that of the SMS397A
strain (57.3 U/ml) grown in GPP medium adjusted to an initial pH of 5.0 (Table 2). During the cultivation of the
CS3 strain, the pH level remained constant at 5.0 while that of the
SMS397A culture increased from 5.0 to 5.7. Glover et al. suggested that
the expression of the AXP gene is regulated by the pH, and
the highest level of AXP mRNA was detected at a pH range of
5.0 to 5.5 (10). It is likely that the lower pH in the CS3
strain slightly enhanced the expression of the AXP gene and
this resulted in the higher enzyme activity in the CS3 strain (92.0 U/ml) compared to that of the SMS397A strain (57.3 U/ml). Western blot
analysis revealed that both the SMS397A and CS3 strains produced and
secreted the same 39-kDa AXP band (Fig.
3). As Glover et al. pointed out that
secreted AXP proenzyme undergoes activation extracellularly by
autocatalytic cleavage at acidic pH, the processing of AXP is
independent of Ca2+ regulation in the Golgi apparatus
(10). These results suggest that the YlPMR1
disruption affects the activity of the secreted AXP at the pH where its
expression is induced, whereas the secretion process of premature AXP
protein is not affected by the YlPMR1 disruption.
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TABLE 2.
Effect of YlPMR1 disruption on the activity of
AXP in Y. lipolytica SMS397A (YlPMR1) and
CS3 (Ylpmr1) strains
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FIG. 3.
Western blot analysis of AXP from SMS397A
(YlPMR1) and CS3 (Ylpmr1) strains grown in GPP
medium at an initial pH of 5.0. Lane 1, supernatant of SMS397A; lane 2, cell extract of SMS397A; lane 3, supernatant of CS3; lane 4, cell
extract of CS3.
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Inhibition of secretion of heterologous rice -amylase.
The
expression and efficient secretion of rice -amylase in Y. lipolytica was previously reported (27). The expression
vector (pXOS103-In) was transformed into SMS397A and CS3 strains to
study the effects of the YlPMR1 disruption on the secretion
of heterologous proteins. Integration of the expression vector into
chromosomal DNA was confirmed by Southern blot analysis
(27). The transformants of both strains were grown on
YM-starch plates, and secretion of rice -amylase was detected by
using iodine vapor. In liquid culture, relatively high rice -amylase
activity (2.75 U/ml) was detected in the transformed SMS397A strain
(SMS-RA), while very low enzyme activity (0.46 U/ml) was observed in
the transformed CS3 strain (CS3-RA) during all stages of growth (Fig.
4b). Although the SMS397A-RA
transformant showed significant -amylase activity, the CS3-RA
transformant showed no clear zone around the colony, indicating the
absence of -amylase activity on the YM-starch plate (Fig. 4a). The
effect of the YlPMR1 disruption on rice -amylase secretion was further confirmed by Western blot analysis. Western blot
analysis revealed that neither extracellular nor intracellular rice
-amylase was detected in the CS3-RA strain cultured in YPD medium
(Fig. 4c, cf. lanes 1 and 2 with lanes 3 and 4). When 10 mM
CaCl2 was added to the YPD medium, secretion of rice
-amylase was partially restored in the CS3-RA strain (Fig. 4c, cf.
lanes 7 and 3), while intracellular rice -amylase was not detected (Fig. 4c, cf. lanes 8 and 4) regardless of the concentration of calcium
added to the medium. These results suggest that secretion of rice
-amylase is almost completely blocked by the YlPMR1
disruption.
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FIG. 4.
Effects of YlPMR1 disruption on the secretion
of rice -amylase in Y. lipolytica. (a) Test of
-amylase activity on a YM-starch plate. 1, SMS-RA; 2, CS3-RA. (b)
Secretion of recombinant rice -amylase in a flask culture (GPP
medium).  , SMS-RA (growth);  , CS3-RA (growth);  , SMS-RA
(activity);  , CS3-RA (activity). (c) Western blot analysis of rice
-amylase from SMS397A-RA (YlPMR1) and CS3-RA
(Ylpmr1) strains grown in YPD medium supplemented with 10 mM
CaCl2. The Western blot experiment was performed with
anti-barley -amylase antibody. Lanes 1 and 5, supernatants of
SMS397A-RA culture grown in low-calcium (~180 µM) and high-calcium
(10 mM) concentration media, respectively; lanes 2 and 6, cell extracts
of SMS397A-RA culture grown in low- and high-calcium concentration
media, respectively; lanes 3 and 7, supernatants of CS3-RA culture
grown in low- and high-calcium concentration media, respectively; lanes
4 and 8, cell extracts of CS3-RA culture grown in low- and high-calcium
concentration media, respectively.
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Since the secretion response of the Ca
2+-ATPase-deleted
S. cerevisiae pmr1 mutant (
29) was found to be
different from that
of the
Y. lipolytica Ylpmr1 mutant, the
rice
-amylase gene was
expressed in
YlPMR1-disrupted
S. cerevisiae (AA274) and its isogenic
wild type (AA255) to
examine whether the effect of secretory pathway
Ca
2+-ATPase
disruption on heterologous protein secretion is species
specific or
protein specific. The rice
-amylase signal sequence
and coding
sequence were inserted into the yeast expression vector,
pDB20, between
the alcohol dehydrogenase promoter and terminator
sequences. The
resulting vector (pSCRA) was transformed into
S. cerevisiae
AA255 and AA274 separately, and secretion of rice
-amylase
was
determined in liquid and solid plate cultures. As shown in
Fig.
5a, the AA255 transformant (AA255-RA)
exhibited rice
-amylase
activity, as indicated by the halo around
the colony, while the
AA274 transformant (AA274-RA) showed no enzyme
activity. These
results demonstrate that disruption of the
YlPMR1 gene also created
an inhibitory effect on secretion
of heterologous rice
-amylase
in
S. cerevisiae. The
secreted
T. reesei EGI in
S. cerevisiae AA255 and
AA274 gave positive responses on an ostrazin brilliant
red
H-3B-conjugated hydroxyethyl cellulose (OBR-HEC)-containing
YM plate
(Fig.
5b). Thus, we can conclude that improvements in
heterologous
protein secretion by the disruption of the secretory
pathway
Ca
2+-ATPase are not applicable to all proteins in
Y. lipolytica and
S. cerevisiae strains.
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FIG. 5.
Effects of PMR1 disruption in S. cerevisiae on the secretion of rice -amylase and T. reesei EGI. (a) -Amylase activity on a YM-starch plate. 1, AA255-RA; 2, AA274-RA. (b) EGI activity on an OBR-HEC-containing YM
plate (25). 1, AA255-FC; 2, AA274-FC.
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Change in outer-chain glycosylation of heterologous fungal
EGI.
The EGI expression vector pXCSIn(myc) was transformed in both
the SMS397A and CS3 strains, and the secretion of recombinant EGI was
detected on solid plate and liquid cultures (Fig.
6). Although the plate assay showed
similar halos around the colonies of the SMS397A and CS3 transformants
(SMS-FC and CS3-FC, respectively) (Fig. 6a), the liquid cultures showed
that SMS-FC secreted more EGI than CS3-FC (Fig. 6b). However, if we
consider the cell concentrations of both strains and the specific
activities of the secreted proteins (196 U/mg of protein for SMS-FC and
176 U/mg of protein for CS3-FC), the amounts of EGI produced by both
strains were almost the same. This result is also consistent with that
shown in Fig. 5b.
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FIG. 6.
Effects of YlPMR1 disruption on the secretion
of T. reesei EGI in Y. lipolytica. (a) EGI
activity on an OBR-HEC-containing YM plate. 1, SMS-FC; 2, CS3-FC. (b)
Secretion of recombinant EGI in a flask culture (GPP medium). ,
SMS-FC (growth); , CS3-FC (growth); , SMS-FC (activity); ,
CS3-FC (activity). (c) Western blot analysis for recombinant EGI. Lane
1, culture supernatant of SMS397A (host cell); lane 2, culture
supernatant of SMS-FC; lane 3, culture supernatant of CS3-FC; and lanes
4 and 5, culture supernatants of SMS-FC and CS3-FC, respectively, after
endo H treatment.
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The secreted EGI was examined by Western blot analysis with an anti-myc
antibody (Fig.
6c). While the recombinant EGI produced
by the SMS-FC
strain was hyperglycosylated, the
YlPMR1-disrupted
strain
displayed a homogeneous single band without any smear corresponding
to
hyperglycosylation. To determine the amounts of N-linked glycosylation
in the secreted proteins, endo-
-
N-acetylglucosaminidase H
(endo
H) treatment was performed. The endo H-treated EGI secreted by
CS3-FC displayed a homogeneous single band around 55 kDa, which
is
smaller than the untreated protein (58 kDa) (Fig.
6c, cf. lanes
4 and 5 with lanes 2 and 3). Endo H cleaves high mannose and some
hybrid
oligosaccharides from the N-linked glycosyl group of glycoproteins.
Although the recombinant EGI secreted from the
YlPMR1-disrupted
strain is not hyperglycosylated compared to
the control strain
(SMS-FC), it contains N-linked glycosyl residues
(possibly core
glycosylation) which can be removed by endo
H.
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DISCUSSION |
This study clearly shows that the secretion patterns of the
YlPMR1-disrupted mutant (CS3) and the isogenic wild-type
strain (SMS397A) are different in Y. lipolytica. Secretion
of homologous AEP is dramatically decreased and premature precursors
that have not been processed completely (52- and 36-kDa proteins) were
secreted by the YlPMR1-disrupted mutant (Fig. 1). Enderlin
and Ogrydziak reported that the Xpr6p (Kex2p-like endoprotease), which
is the primary protease required for Lys-Arg cleavage of AEP, is
Ca2+ dependent and that mutation of XPR6 causes
formation of the 52-kDa precursor (8). Based on these data,
we can assume that disruption of the YlPMR1 gene causes the
Ca2+ depletion in the Golgi apparatus and thereby affects
the function of the Ca2+-dependent Xpr6p and results in the
secretion of premature precursors of AEP. When 10 mM calcium was added
to the YPD medium, the secretion of AEP was partially restored (Fig.
2). However, Western blot analysis with the AEP antibody in the
YlPMR1-disrupted mutant showed two bands (36 and 32 kDa)
while only a 32-kDa band was observed for the control strain. It has
been suggested that the 36-kDa polypeptide is a possible precursor for
the mature AEP (22).
It has been reported that the SMS397A strain displays a dimorphic
characteristic (a mixture of mycelia and oval-shaped cells) while the
CS3 strain shows clustered strings of bead-like cells in a GPP medium
(26). The morphology of the CS3 strain is similar to that of
the XPR6-defective mutant. This is additional evidence for
our suggestion that the YlPMR1 gene disruption affects the function of Xpr6p in the CS3 mutant strain.
The findings that the AXP activity of the CS3 strain was significantly
increased (60%) and the AEP secretion by SMS397A was inhibited at pH
5.0 are closely related to the perturbation of calcium concentration
and culture pH. During cultivation, the CS3 strain remained constant at
pH 5.0, but the SMS397A culture pH increased from 5.0 to 5.7 (Table 2).
Disruption of the YlPMR1 gene caused the difference in the
culture pH patterns of these two strains.
While rice -amylase is efficiently secreted by the SMS-RA strain,
very low levels of rice -amylase secretion were observed for the
CS3-RA strain (Fig. 4b). An inhibitory effect of a
Ca2+-ATPase deletion in the S. cerevisiae
PMR1-disrupted strain on the secretion of rice -amylase was
also observed (Fig. 5a). Our results show that recombinant rice
-amylase neither secretes into the culture medium nor accumulates in
the YlPMR1-disrupted mutant cells and that an addition of
exogeneous Ca2+ can partially recover the secretion of rice
-amylase (Fig. 4c). Jones et al. determined the effect of calcium on
the secretion of -amylase and other hydrolases from aleurone layers
of barley (18). They observed that withdrawal of
Ca2+ from the incubation medium of aleurone layers resulted
in a 70 to 80% reduction of -amylase secretion. Also, Bush et al.
showed that barley -amylase is irreversibly inactivated by the
removal of Ca2+ from the protein and that Ca2+
stabilizes the tertiary structure of the enzyme (6). They have also shown that millimolar levels of calcium are necessary to
stabilize barley -amylase in the ER of the aleurone layer. Since
rice -amylase is highly homologous to cereal -amylases including
barley -amylase, it is speculated that production of recombinant
rice -amylase in CS3-RA cells is almost completely inhibited by the
intracellular Ca2+ deficiency which results from the
YlPMR1 disruptions in Y. lipolytica.
Unlike rice -amylase, the secretion of T. reesei EGI was
not influenced by the YlPMR1 disruption. However, the
secreted EGI from the YlPMR1-disrupted mutant did have
different characteristics than that of the control (Fig. 6). While
wild-type cells secreted the hyperglycosylated form of EGI,
hyperglycosylation was completely absent from the mutant strain. This
result confirms that disruption of the secretory pathway
Ca2+-ATPase causes disrupted outer-chain glycosylation of
secreted proteins in yeast (29). However, endo H treatment
of the secreted EGI shows that there are some glycosyl groups at the
N-glycosylation site, indicating only a partially defective outer-chain
glycosylation in the YlPMR1-disrupted mutant.
In this study, the effects of disrupted secretory pathway
Ca2+-ATPase in the yeast Y. lipolytica on the
secretion and posttranslational processing of homologous and
heterologous proteins were examined by using AEP, AXP, rice
-amylase, and fungal EGI as model proteins. In the
YlPMR1-disrupted mutant, secretion of mature AEP decreased while the premature 36- and 52-kDa AEPs were secreted and intracellular 52-kDa premature AEP was also detected. Production of rice -amylase was completely blocked in the YlPMR1-disrupted mutant.
Production and secretion of recombinant fungal EGI was not affected,
while hyperglycosylation of EGI was removed. Production of AXP was
increased by the YlPMR1 disruption. Our results indicate
that the effects of the YlPMR1 disruption depend on the
characteristics of the target protein in the recombinant yeast strain evaluated.
| |
ACKNOWLEDGMENTS |
This research was partially supported by grants from the National
Science Foundation and the University of California Systemwide Biotechnology Research and Education Program.
We thank David Ogrydziak for his helpful discussions and for sharing
the anti-AEP antibody and strain SMS397A, G. R. Fink for sharing
the S. cerevisiae PMR1-disrupted mutant (AA274) and its
isogenic wild-type strain (AA255), S. Katoh and M. Terashima for
sharing anti-barley -amylase antibody, T. W. Young for anti-AXP antibody, and Mike Ward for pEndo of T. reesei EGI.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biochemical
Engineering Program, Department of Chemical Engineering and Material
Science, University of California, One Shields Avenue, Davis, CA 95616. Phone: (530) 752-8954. Fax: (530) 752-3112. E-mail:
ddyryu{at}ucdavis.edu.
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Journal of Bacteriology, December 1998, p. 6736-6742, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
