World J Microbiol Biotechnol (2017) 33:210
DOI 10.1007/s11274-017-2361-z
REVIEW
Saccharomyces cerevisiae proteinase A excretion and wine making
Lulu Song1 · Yefu Chen1 · Yongjing Du1 · Xibin Wang1 · Xuewu Guo1 · Jian Dong1 ·
Dongguang Xiao1 
Received: 2 August 2017 / Accepted: 26 September 2017 / Published online: 9 November 2017
© Springer Science+Business Media B.V. 2017
Abstract  Proteinase A (PrA), the major protease in Sac-
charomyces cerevisiae, plays an essential role in zymogen
activation, sporulation, and other physiological processes
in vivo. The extracellular secretion of PrA often occurs dur-
ing alcoholic fermentation, especially in the later stages
when the yeast cells are under stress conditions, and affects
the quality and safety of fermented products. Thus, the
mechanism underlying PrA excretion must be explored to
improve the quality and safety of fermented products. This
paper briefly introduces the structure and physiological
function of PrA. Two transport routes of PrA, namely, the
Golgi-to-vacuole pathway and the constitutive Golgi-to-
plasma membrane pathway, are also discussed. Moreover,
the research history and developments on the mechanism
of extracellular PrA secretion are described. In addition,
it is briefly discussed that calcium homeostasis plays an
important role in the secretory pathway of proteins, imply-
ing that the regulation of PrA delivery to the plasma mem-
brane requires the involvement of calcium ion. Finally, this
review focuses on the effects of PrA excretion on wine mak-
ing (including Chinese rice wine, grape wine, and beer brew-
age) and presents strategies to control PrA excretion.
Keywords  Proteinase A · Golgi-to-vacuole pathway ·
Constitutive Golgi-to-plasma membrane pathway ·
Calcium homeostasis · Wine making · Saccharomyces
cerevisiae
* Yefu Chen
yfchen@tust.edu.cn
1
Key Laboratory of Industrial Fermentation Microbiology,
Ministry of Education, Tianjin Industrial Microbiology
Key Laboratory, College of Biotechnology, Tianjin
University of Science and Technology, Tianjin 300457,
People’s Republic of China
Introduction
Saccharomyces cerevisiae PrA (saccharopepsin; EC
3.4.23.25) is encoded by the PEP4 gene, which is located
proximal to GAL4 on chromosome XVI (Ammerer et al.
1986; Parr et al. 2007; Woolford et al. 1996). PrA is nor-
mally exported from the Golgi apparatus to the vacuole,
whereas PrA is released from living yeasts cells under stress
conditions (Kondo et al. 1999). PrA is essential to the vacu-
olar proteolytic system under various growth conditions,
including nutritional stress, sporulation, and vegetative
growth conditions (Hirsch et al. 1992; Jones 1984). This
protease also plays an important role in the maturity of other
hydrolases, such as carboxypeptidase (CPY) and proteinase
B (PrB) (Parr et al. 2007). The excreted PrA significantly
affects the safety and quality of alcoholic drinks, especially
the foam stability of beer (Zhang et al. 2011). Thus, many
works were conducted to reduce PrA secretion and improve
the safety and quality of alcoholic drinks.
In this paper, we summarize current knowledge regarding
the structure and function of PrA, the two transport routes
of PrA, and the effects of calcium homeostasis on protein
transport. This review also focuses on the effects of PrA
excretion on wine making and the approaches of decreasing
PrA excretion.
Structure and physiological function of PrA
Structure of PrA
The amino acid sequence of PrA is first reported by Dreyer
et al. (1986). PrA is synthesized as an inactive precursor of
405 amino acids (Parr et al. 2007). The inactive preform of
PrA, designated as preproPrA, consists of an amino-terminal
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signal peptide (1–22 residues), propeptide (23–76 residues),
and mature PrA (77–405 residues) (Fig. 1) (Klionsky et al.
1988).
The mature PrA possesses primary, secondary, and ter-
tiary structures (Table 1), which have been widely reported.
The N-terminal 76 amino acids of preproPrA, which con-
tain an amino-terminal signal peptide and propeptide, are
cleaved in the endoplasmic reticulum (ER) and the vacuole,
respectively (Fig. 1), to generate the mature 329-amino-acid
enzyme; in this enzyme, 43% are polar residues, and 12%
are aromatic residues (Dreyer et al. 1986; Klionsky et al.
1988; Meussdoerffer et al. 1980). After glycosylation in the
ER, the mature PrA contains additional 1% glucosamine and
7.5% neutral sugars, with carbohydrate moieties attached at
Asn267 and Asn67 (Dreyer et al. 1986; Jones 1984; Meuss-
doerffer et al. 1980). All these descriptions are primary
structural characteristics of S. cerevisiae PrA.
S. cerevisiae PrA is an aspartic proteinase, and its sec-
ondary and tertiary structures are similar to those of other
aspartic proteinase. PrA consists largely of β-sheets; the
secondary structure of PrA displays ten small right-handed
α-helical segments and some hairpins (Aguilar et al. 1997).
Fig. 1  Schematic representation of the PEP4 gene product (termed
preproPrA), and processing site of preproPrA in the ER and the vacu-
ole (van den Hazel et al. 1993; Wolff et al. 1996). The open, hatched
and solid bars indicate an amino-terminal signal peptide (prepeptide),
propeptide, and mature PrA, respectively. The arrows indicates the
cleavage sites of preproPrA
Table 1  The brief description
of mature S. cerevisiae PrA
structure The primary structure
The secondary structure
The tertiary structure
a
 Compared with α-helical and hairpin, the contents of β-sheet are relatively higher
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World J Microbiol Biotechnol (2017) 33:210
Information on the tertiary structure of PrA is present in Pro-
tein Data Bank (http://www.rcsb.org/pdb/explore/explore.
do?structureId=2JXR). PrA contains two topologically simi-
lar lobes, each of which provides one catalytic aspartic acid
residue; the two lobes converge in the plane of a two-fold
axis of symmetry (Parr et al. 2007). Two disulfide bonds
(located in residues 45–50 and 249–282) make the tertiary
structure of PrA stable (Aguilar et al. 1997; Parr et al. 2007).
Additionally, PrA exhibits a flexible “flap”, which consists
of a β-hairpin loop extending over the active site (Parr et al.
2007). Tyr75, a conserved residue, is located at the tip of
the flap and involved in the capture and cleavage of sub-
strates. Nevertheless, the unusual orientation of Tyr75 may
exert a negative effect on PrA catalysis (i.e., self-inhibition)
(Gustchina et al. 2002; Parr et al. 2007).
Physiological function of PrA
Saccharomyces cerevisiae PrA is located in the acidic
vacuole (Hata et al. 1967; Lenney and Dalbec 1967). PrA
is considered to be involved in intracellular proteolysis at
acidic pH and inhibited by two aspartic proteinase inhibi-
tors, namely, 1,2-epoxy-3-(p-nitrophenoxy) propane and
diazoacetyl-d,l-norleucine methyl ester (Lundblad and Stein
1969; Tang 1971). On the basis of its proteolytic activity
and inhibition, PrA is classified as an aspartic proteinase
(Dreyer et al. 1986). PrA possesses two aspartic acid resi-
dues, namely, A­ sp32-Thr-Gly and A­ sp215-Thr-Gly, which are
required for catalytic action (Kondo et al. 1998). PrA also
plays important roles in a number of physiological and bio-
chemical processes of yeast cells.
PrA is required for unspecific proteolytic processes, such
as nitrogen starvation-induced sporulation-associated pro-
tein degradation (Jones 1984). PrA-induced protein pro-
teolytic processes provide amino acid for synthesis of new
proteins under nutritional stress condition (Teichert et al.
1989). In yeast mutants lacking proteinase A, sporulation
frequency is decreased, due to the disturbing of differen-
tiation process of sporulation (Mechler and Wolf 1981).
Description References
43% polar residues Dreyer et al. (1986)
12% aromatic residues Jones (1984)
7.5% neutral sugars Klionsky el al. (1988)
1% glucosamine Meussdoerffer et al. (1980)
β-sheeta Aguilar et al. (1997)
Right-handed α-helical
Hairpin
Two topologically similar lobes Aguilar et al. (1997)
Two disulfide bonds Parr et al. (2007)
A flexible “flap”
World J Microbiol Biotechnol (2017) 33:210 Page 3 of 12  210

Nonetheless, any highly specific proteolytic process is not
required for the involvement of PrA (Jones 1984). An exam-
ple that approves of this opinion is as follows: Mechler and
Wolf (1981) reported that activation or inactivation of vital
proteins required for the vegetative cell cycle is not affected
by lacking PrA. Moreover, the carbon starvation-induced
inactivation of the NADP-dependent glutamate dehydroge-
nase and the activation of chitin synthase zymogen, which
are proposed as proteolytic events, are not influenced with
the absence of PrA (Jones 1984).
In addition to the activation of immature PrA, S. cerevi-
siae PrA is also responsible for the activation process of
other vacuolar hydrolases, including PrB and CPY (Parr
et al. 2007; van den Hazel et al. 1992). The propeptides
of PrB and CPY are directly cleaved by the mature PrA to
yield mature enzymes (Ammerer et al. 1986; van den Hazel
et al. 1993). The vacuolar hydrolytic activities of PrA are
implicated in the activation of trehalase, RNase, alkaline
phosphatase, and aminopeptidase I (Harris and Cotter 1987;
Klionsky et al. 1992; van den Hazel et al. 1992; Zubenko
et al. 1982). Vacuolar PrA may also control the glycolytic
enzyme expression in direct or indirect manner, such as the
expression of phosphofructokinase, hexokinase, and pyru-
vate kinase. Thus, the direction and rate of glycolytic flux
may be regulated by vacuolar mature PrA (Chen et al. 2010).
Additionally, propeptide contributes to the folding of the
mature PrA region into its stable active conformation and
functions as a vacuolar protein sorting signal (Dreyer et al.
1983; Saheki et al. 1974). Although the current knowledge
about the physiological function of PrA is extensive (sum-
marized in Table 2), the information is still incomplete.
Two transport routes for PrA
Vacuolar sorting pathway of PrA
The sorting and transport of intracellular and extracel-
lular proteins are mediated with the secretory pathway.
Many organelles are included in the secretory pathway.
Most proteases, which will be delivered to the vacuole,
pass through many organelles on the way to the vacuole.
The transport between two organelles is mediated by vesi-
cles. S. cerevisiae PrA is a vacuolar protease (van den
Hazel et al. 1996). After manufacturing on ribosomes, PrA
passes through the ER and the Golgi complex, and it is
subsequently delivered to the vacuole.
PrA is encoded as a preproenzyme by the PEP4 gene
on the ribosomes (Wolff et al. 1996). The N-terminus of
preproPrA contains a hydrophobic signal peptide (Klion-
sky et al. 1988). The hydrophobic signal peptide promotes
the translocation of preproPrA into the ER (Klionsky et al.
1988; van den Hazel et al. 1996). A possible reason for
this conclusion is that the hydrophobic signal peptide is
recognized by the signal-recognition particle (SRP), and
the complex is subsequently targeted to the SRP receptor
at ER translocation sites (Barlowe and Miller 2013). Upon
arrival in the lumen of ER, 22 amino acid residues at the
N-terminus of preproPrA are cleaved off, and preproPrA
is glycosylated at Asn67 and Asn267 (Dreyer et al. 1986).
The glycosylated precursors are packaged into vesicles and
translocated into the Golgi complex (Pryer et al. 1992). In
the lumen of Golgi apparatus, the carbohydrate side chains
of glycosylated precursors undergo limited extension with
α-1→3-mannose linkages to produce a high-molecular-
weight form (proPrA) of 52 kDa (Klionsky et al. 1988).
Finally, proPrA is sorted to the vacuole from the trans-
Golgi network (TGN). Upon arrival in the vacuole, the
PrA propeptide is removed, and the active, mature PrA
(42 kDa) is generated by the specific proteolysis of PrB
or autoactivation (Klionsky et al. 1988; Parr et al. 2007;
van den Hazel et al. 1992). Golgi apparatus contains com-
plex structure, and it is consisted of cis-Golgi network,
medial-Golgi network, and TGN (Pryer et al. 1992). The
structural complexity implies multiple functions, includ-
ing protein maturation, recognition, and sorting, occurring
in this organelle. A large number of sorting events occur
in the TGN (Pryer et al. 1992). ProPrA is sorted to the
vacuole from the TGN via the Golgi-to-vacuole pathway
(Fig. 2a upper panel).

Table 2  Summary of representative examples regarding S. cerevisiae PrA function

Examples References

Proteolysis of mature PrA Degradation of population-associated protein Jones (1984)

Autoactivation Harris and Cotter (1987)
Activation of mature PrA on other hydrolases The activation of PrB and CPY Klionsky et al. (1992)
Implicated in implicated in the activation of trehalase, Parr et al. (2007)
RNase, alkaline phosphatase, and aminopeptidase I van den Hazel et al. (1992)
Zubenko et al. (1982)
The other functions of mature PrA Implicated in glycolytic enzymes expression Chen et al. (2010)
Propeptide of PrA Protein folding; Klionsky et al. (1988)
Vacuole targeting van den Hazel et al. (1993)

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Fig. 2  Two secretory pathways of PrA (Chen et  al. 2017; Kondo
et  al. 1999). The upper panel of a shows the Golgi-to-vacuole path-
way of PrA; the lower panel of a shows the default pathway of PrA
Westphal et al. (1996) reported that the sorting of proPrA
to the vacuole from the TGN is a vacuolar sorting receptor
(VSR)-mediated process. Several VSRs are involved in the
sorting process of proPrA from the TGN to the vacuole, and
the most remarkable typical receptor is Vps10p (type I trans-
membrane receptor protein) (Cooper and Stevens 1996; Jør-
gensen et al. 1999; Marcusson et al. 1994). Previous research
originally discovered that Vps10p is a sorting receptor for
transporting CPY to the vacuole (Marcusson et al. 1994).
Analogous to CPY, Vps10p functions as a sorting receptor
for proPrA (Cooper and Stevens 1996; Chen et al. 2017).
In the TGN of mammalian cells, the mannose-6-phosphate
receptor (M6PR), which is required for the export of lyso-
somal hydrolases to the lysosomes, binds mannose-6-phos-
phate-containing lysosomal hydrolases (Baranski et  al.
1990). Mrl1p, a homolog of M6PR, exists in the yeast cells
and contributes to the sorting of PrA (Whyte and Munro
2001). Whyte and Munro (2001) previously reported that
20% correctly mature PrA is transported to the vacuole by
deleting both MRL1 and VPS10. These findings implied that
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World J Microbiol Biotechnol (2017) 33:210
excretion, which may be consistent with the constitutive Golgi-to-
plasma membrane pathway shown in the b 
in addition to Vps10p and Mrl1p, other VSRs are involved in
PrA sorting. The overexpression of Vth1p and Vth2p, which
are highly homologous to Vpsl0p, suppresses the missorting
phenotypes of CPY and PrA in the mutant lacking Vpsl0p
(Cooper and Stevens 1996; Westphal et al. 1996); this phe-
nomenon indicates that Vth1p and Vth2p may act as a VSR
of PrA. However, PrA sorting is not affected by the disrup-
tion of Vth1p and Vth2p (Cooper and Stevens 1996). These
results indicate that Vth1p and Vth2p play a minor role in
the receptor-mediated sorting process of PrA. Furthermore,
there are at least three mechanisms of VSR-mediated sort-
ing, namely, Vps10p-dependent path, Mrl1p-dependent path,
and Vps10p/Mrl1p/Vth1p/Vth2p-independent path. How-
ever, the Vps10p/Mrl1p/Vth1p/Vth2p-independent pathway
remains unknown to date.
The corresponding receptor recognition sites, which bind
the VSRs, are required for the sorting of proPrA to the vacu-
ole. The hybrid protein, which consists of the 54-amino-
acid propeptide of PrA and intact periplasmic enzyme
invertase, is sorted to the vacuole, which indicated that the
World J Microbiol Biotechnol (2017) 33:210 Page 5 of 12  210
PrA propeptide contains the vacuolar sorting information
(Klionsky et al. 1988). The PrA propeptide contains the sort-
ing information, and it is recognized by Vps10p (Westphal
et al. 1996). Consequently, Vps10p recognizes and binds
proPrA, and it is packed into clathrin-coated vesicles for
transport to the vacuole. Not all the VSRs sort PrA to the
vacuole through a peptide-based interaction. For the M6RP
of mammalian cells, the yeast Mrl1p affects the sorting
of PrA to the vacuole through a glycan-based interaction
(Whyte and Munro 2001). In addition, the PrA propeptide is
essential for PrA folding and translocation into the ER (van
den Hazel et al. 1993). Mature PrA contains vacuole sorting
signals (van den Hazel et al. 1995).
Extracellular secretory pathway of PrA under stress
conditions
PrA is generally considered not released from the yeast cells.
PrA is also detected in the extracellular medium, which is
thought to be caused by the yeast cell autolysis. Neverthe-
less, Maddox and Hough (1970) reported that proteolytic
enzymes are secreted from the living yeast cells when casein
is used as sole nitrogen of the yeasts. PrA may be excreted
by the living yeasts, which is similar to those of proteolytic
enzymes. Dreyer et al. (1983) indicated that the living cells
excrete PrA to the medium. Result confirmed that the phe-
nomenon of PrA excretion in the living yeasts occurs. To
explain this phenomenon, the mechanism of PrA excretion
from living yeast cells is extensively studied.
A large number of stress proteins are generated in S. cer-
evisiae cells; these proteins respond to a variety of adverse
environmental conditions (Sanchez et al. 1992). Gibson
et al. (2007) accounted that 200 genes and the correspond-
ing proteins are up-regulated in the general stress response
(GSR). During wine making, their expression levels of
numerous genes are changed to adapt to the fermentation of
grape must; 233 of these genes are involved in response to
fermentation stress (Marks et al. 2008). Whether PrA excre-
tion is a response to adverse extracellular environment is
unclear. Previous reports indicated that the level of intracel-
lular PrA is related to the contents of carbon and nitrogen.
Specifically, the yeast cells, which grow on poor carbon or
nitrogen sources, result in several folds of increases in the
activities of PrA (Hansen et al. 1977). Saheki and Holzer
(1975) described that the PrA activities increased several
times during transition from logarithmic phase to station-
ary phase. This finding could be due to the lower glucose
content in the medium of stationary phase than that in
the logarithmic growth phase, resulting in carbon source-
stressed condition. PrA, together with PrB, is responsible
for approximately 40% of protein degradation under veg-
etative growth conditions. Nonetheless, their participation
in protein degradation is more than 80% under nutritional
stress (Teichert et al. 1989). These findings implied that PrA
may be a stress protein, and its production is considerably
enhanced to respond to nutritionally stressed conditions and
degrade stress proteins. Therefore, PrA excretion may be a
behavior of the GSR. The mechanism of how PrA is excreted
into the medium is also unknown.
Kondo et al. (1999) deduced a hypothetical pathway of
PrA excretion from the living yeast cells (Fig. 2a lower
panel). PrA is mistakenly excreted from the living yeast cells
under stress conditions, rather than being precisely trans-
ported to the vacuole from the Golgi complex as normal
metabolism. ProPrA, which is released from the cells, is
converted to the mature PrA by autocatalytic processing in
the medium. However, the trigger mechanisms of the extra-
cellular secretory of PrA remain unknown. PrA is misdi-
rected to the cell surface and subsequently secreted to the
medium due to overproduction of PrA (Rothman et al. 1986;
Wolff et al. 1996). Moreover, PrA overproduction results in
saturation of a step in the vacuolar sorting process (Rothman
et al. 1986). Disruption of one or more allele of the PEP4
gene decreases the extracellular PrA activities to varying
degrees (Lu et al. 2012; Wang et al. 2007a, b). The expres-
sion level of PrA is an influencing factor that triggers PrA
excretion. Disrupting the prosequence of PrA (encoding the
proteinase A propeptide) decreases the extracellular activ-
ity of PrA (Wu et al. 2016). This result can be explained by
that the propeptide disruption may lead to the degradation of
proPrA in the ER, which lowers the intracellular PrA activity
(van den Hazel et al. 1993). Thus, the sorting signal strength
of PrA may be also important for the trigger mechanisms
of PrA excretion. The yeast cells lacking Vpsl0p missort a
large number of PrA to the cell surface (Cooper and Stevens
1996; Marcusson et al. 1994). The extracellular activity of
PrA is decreased in the Vpsl0p overexpression mutant (Chen
et al. 2017). The VSRs may be a key factor that triggers PrA
excretion.
In mammalian cells, the secreted proteins are transported
to the cell surface via either of the two pathways: the consti-
tutive secretory pathway (CSP) and the regulated secretory
pathway (RSP) (Brion et al. 1992; Burgess and Kelly 1987;
Kelly 1985). When the vacuolar sorting system is saturated,
or the sorting signal is unreadable or missing, proteins are
secreted from the living yeast cells via a default pathway,
namely, the constitutive Golgi-to-plasma membrane path-
way (CGPMP), which is similar to the CSP of mammalian
cells (Burgess and Kelly 1987; Kelly 1985). The delivery
of secreted protein via the CSP requires no involvement of
the protein sorting signal; this delivery is also a nonspecific,
bulk flow process (Burgess and Kelly 1987; Kelly 1985).
Overproduction and the N-terminus deletion of the vacu-
olar protein procarboxypeptidase Y (proCPY) result in its
aberrant secretion, and the aberrant secretion of proCPY can
be blocked by inhibiting the CSP (Burgess and Kelly 1987;
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Page 6 of 12
Stevens et al. 1986; Valls et al. 1987). Burgess and Kely
(1987) concluded that missorted CPY is secreted from the
cells via CSP. Hence, mislocalized PrA is probably delivered
to the cell surface via CGPMP as CPY (Fig. 2b). The trans-
port events, that is, secreted proteins directly reach the cell
surface after the processing of the Golgi apparatus, are medi-
ated by secretory vesicles (SV). SVs release their contents
by exocytosis (Burgess and Kelly 1987). Exocytosis may
consist of four essential steps: vesicle budding, transport,
tethering, and fusion (Bonifacino and Glick 2004). GTPase
Ypt31/32 is recruited to the TGN membrane and is correctly
assembled with coat proteins to form buds (Benli et al. 1996;
Springer et al. 1999). Afterward, the SV-Ypt31/32 complex
containing targeted proteins leaves the TGN. After being
activated by Sec2 (guanine nucleotide exchange factor),
Sec4 replaces Ypt31/32, binds to SV, and interacts with
exocyst; consequently, the tethering process is activated
(Bröcker et al. 2010; Liu and Guo 2012; Ortiz et al. 2002).
v-SNARE and t-SNARE are located in the SV and plasma
membrane, respectively (Hong and Lev 2014). The tether-
ing process promotes the binding of v-SNARE to t-SNARE,
which results in the fusion of SV with plasma membrane
(Söllner et al. 1993). The absence of Sec5p, a core subunit
of the exocyst, remarkably reduces the secretion of PrA from
the living cells via CGPMP (Chen et al. 2017; Terbush and
Novick 1995). These results are consistent with the hypoth-
esis of blocking the CGPMP of PrA.
Regulation of calcium homeostasis in protein
transport
In addition to their functions in post-translational modifica-
tion, sorting, and translocation of vacuolar, secreted, and
membrane proteins, the organelles of the secretory pathway
exhibit a pivotal role in maintaining the cellular calcium
homeostasis. Processes occurring in the secretory pathway
of eukaryotes require the involvement of calcium (Curwin
et al. 2012; Fokina et al. 2015). The regulated mechanism of
calcium homeostasis in protein transit and the mechanism of
organelles in supplying calcium ion for the secretory path-
way are mainly discussed in this section.
According to Sambrook (1990), the transport of secre-
tory proteins from the ER requires calcium. The ER is an
intracellular ­Ca2+ reservoir, and the release of C
­ a2+ from the
ER is mediated with inositol-1,4,5-trisphosphate (IP3) and
caffeine (Somlyo et al. 1985; Streb et al. 1984). Perturbation
in lumenal calcium levels in the ER causes breakdown in
resident ER protein sorting system (Booth and Koch 1989).
The possible reason may be that the cycle of resident ER
proteins between the ER and the Golgi complex is a cal-
cium-mediated process, which is interfered with perturbed
calcium contents; consequently, the resident ER proteins are
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World J Microbiol Biotechnol (2017) 33:210
excreted (Booth and Koch 1989). Perturbation of lumenal
calcium levels in the ER blocks the exit of secretory proteins
from the ER and accelerates protein degradation (Lodish and
Kong 1990; Wileman et al. 1991). This result is attributed
to that the protein folding process in the ER is calcium-
dependent; thus, low calcium levels in the ER results in the
corresponding unfolded proteins, which causes the above
phenotype (Lodish and Kong 1990; Wileman et al. 1991).
Considerable evidence showed that Golgi calcium plays a
critical role in the exocytic or secretory pathway. For exam-
ple, proteolytic processing of protein precursors in the Golgi/
TGN is blocked under calcium depletion (Oda 1992). The
­Ca2+ contents in the millimolar range are required for the
activity of many resident Golgi enzymes, including KEX2
endoprotease and guanosine diphosphatase in yeasts (Abei-
jon et al. 1989; Fuller et al. 1989). Maintaining calcium
homeostasis within the lumen of ER/Golgi is critical to the
correct folding, processing, and transit of proteins during
their transportation via the secretory pathway. Nevertheless,
the mechanism of organelles in providing calcium for the
secretory pathway remains unknown.
Ion pump Pmr1p supplies C ­ a2+ to the yeast secretory
pathway, and the secretory pathway is interfered with muta-
tions in PMR1 (Dürr et  al. 1998; Rudolph et  al. 1989).
Antebi and Fink (1992) reported that the Pmr1p located in
the Golgi apparatus transports C ­ a2+ into the Golgi appa-
ratus, and the mutants of Pmr1p function disruption pro-
duce incompletely glycosylated invertase (lacking the outer
chain branched mannose residues) and impair the proteo-
lytic processing of pro-α factor. These studies indicated that
the delivery of C ­ a2+ to the secretory pathway is a Pmr1p-
dependent route. Pmr1p inactivation leads to C ­ a2+ deficiency
in the secretory pathway, and the phenotype is exacerbated
in the three events: (1) the ret1-27 mutation, which influ-
ences the COPI-mediated vesicular transport; (2) inactiva-
tion of Pmc1p (a vacuolar ­Ca2+ ATPase); and (3) inactiva-
tion of Vps35, a component of the retromer complex that
plays a role in trafficking between the vacuole and secretory
organelles (Fokina et al. 2015). On the basis of these results,
another Pmr1-independent route of the ­Ca2+ transportation
to the secretory pathway exists.
The TGN is a secretory protein-sorting station, and
proteins must be correctly sorted for delivery to the tar-
geted destination in the TGN (Traub and Kornfeld 1997).
Recent studies showed that protein sorting from the TGN
also requires calcium (Kienzle and Von Blume 2014). In
mammalian cells, actin-severing protein ADF/cofilin and
the calcium ATPase 1 (SPCA1) are required for the sorting
of secretory protein at the TGN (von Blume et al. 2009,
2011). The cell wall protein Bgl2 is retained in a late Golgi
compartment due to the reduced secretion rate, and CPY
is secreted in the cofilin mutant and the mutant lacking
Pmr1p (the yeast ortholog of SPCA1); these observations
World J Microbiol Biotechnol (2017) 33:210 Page 7 of 12  210
highlighted the conserved role of cofilin and Pmr1p in secre-
tory protein sorting at the TGN in yeast cells, which is simi-
lar to that in mammalian cells (Curwin et al. 2012). In terms
of PrA, at least two exit routes within the Golgi apparatus are
required for its export from the Golgi apparatus to the vacu-
ole or the cell surface. Although the sorting of PrA at the
TGN for the vacuole delivery is a receptor-mediated process,
the corresponding sorting mechanism by which secreted
PrA is sorted for transport to the plasma membrane remains
poorly understood. These findings revealed that the sort-
ing of excreted PrA requires the involvement of cofilin and
Pmr1p. Additionally, the organelles (the ER and the Golgi
apparatus) supply ­Ca2+ for PrA secretory pathway through
Pmr1p-dependent or Pmr1p-independent route.
Effects of PrA excretion on wine making
and product quality
Winemaking (including Chinese rice wine, grape wine, and
beer brewage) is a technique with a long history, and yeasts
play a leading role in the complex process of winemaking
(Pretorius 2000). However, in all stages of different win-
emaking processes, S. cerevisiae strain must encounter vari-
ous stress conditions. PrA is secreted from the living yeast
cells in stress conditions. PrA excretion in the fermentation
broth will affect the quality of alcoholic beverages. This
review discusses the effects of PrA excretion on the quality
and safety of three traditional alcoholic beverages (Chinese
rice wine, wine, and beer).
Yeasts are used to produce brewed wine, including Chi-
nese rice wine, beer, and wine, and they are vital for the
formation of the characteristic flavor compounds and etha-
nol (Chen and Xu 2010). Nonetheless, toxic by-products,
including biogenic amines (BAs) and ethyl carbamate (EC),
which are potentially harmful for human health, may be pro-
duced by S. cerevisiae (Zhao et al. 2014; Zhong et al. 2012).
Both BAs and EC can be widely found in Chinese rice wine,
beer, and wine; the concentration of BAs and EC is generally
lower in beer and grape wine than that in Chinese rice wine
(Zhong et al. 2012; Weber and Sharypov 2009). Decreasing
the BA and EC contents in Chinese rice wine and grape
wine is extensively investigated. However, the effect of PrA
excretion on the concentration of BAs and EC in different
kinds of wine is poorly known.
BAs are a group of organic nitrogenous compounds with
low molecular weight (Lee et al. 2015; Smit et al. 2008).
Trace BAs exhibit metabolic and physiological roles in bio-
logical cells, and excessive intake of BAs could result in
several health problems, including blushing, itching, head-
ache, and skin irritation (Guo et al. 2015a, b). Three prereq-
uisites for the formation of BAs are as follows: (1) the pres-
ence of available free amino acids; (2) microorganisms with
decarboxylase-positive activity; and (3) suitable medium
conditions for these microorganisms (Peña-Gallego et al.
2012). BAs are mainly synthesized by decarboxylation of
their precursor amino acids via specific decarboxylase activ-
ity in different stages of wine production and storage (Guo
et al. 2015a, b; Zhong et al. 2012). As a proteolytic enzyme,
excreted PrA may catalyze the proteolysis of protein in the
medium, thereby resulting in abundant amino acids in dif-
ferent kinds of wine (Teichert et al. 1989); these amino
acids provide the amino acid precursors for BA formation.
Thus, the release of PrA into the medium is implicated in the
concentration of BAs in alcoholic beverages. Recent report
indicated that the concentration of BAs in the Chinese rice
wine is decreased by 25.5% from 180.1 mg/L to 134.2 mg/L
when the PEP4 gene of the yeasts is knocked out (Guo et al.
2015a, b).
EC is the ethyl ester of carbamic acid (Lee 2013; Weber
and Sharypov 2009). EC is previously used as a hypnotic
and an antineoplastic agent for medicine purposes and a
cosolvent for drug manufacture (Zhao et al. 2013). Further-
more, EC is used in the textile industry as chemical interme-
diate to impart wash and wear (Weber and Sharypov 2009).
However, EC is classified as a group 2A carcinogen, which
exerts a potential carcinogenic risk to human (Lachenmeier
2007). At least five available EC precursors, including urea,
carbamyl phosphate, citrulline, diethyl, and cyanogen, can
be converted to EC (Zhao et al. 2013). In most of alcoholic
drinks, urea, mainly accumulated by yeast, is the primary
EC precursor (Zhao et al. 2013). Urea is converted into EC
by reaction with ethanol (Bruno et al. 2007; Riffkin et al.
1989). Urea can be found in raw materials or produced by
yeast as a by-product in arginine catabolism (Schehl et al.
2007; Weber and Sharypov 2009). Proteins in the medium
may be converted into arginine through the proteolysis of
excreted PrA. Evidently, the increasing arginine content in
the medium directly increases the urea yield. Consequently,
the Chinese rice wine and grape wine contain high concen-
tration of EC.
In addition, as a proteolytic enzyme, excreted PrA pro-
motes the proteolysis of proteins into free amino acid,
thereby resulting in abundant amino acids in the Chinese
rice wine and grape wine, namely, high nutritional value.
The aggregation of wine proteins in grape wine causes
flocculation into a hazy suspension and the formation of
precipitates, which affect the clarity of grape wine (Waters
et al. 2010). Excreted PrA may degrade grape wine protein,
influence the formation of haze and precipitates, and finally
improve the sensory quality of the grape wine.
Studies on the relation between beer foam stability and
PrA are relatively thorough. Several beer proteins and poly-
peptides, such as lipid transfer protein 1 (LTP1), protein Z,
and various hordein-derived polypeptides, are required for
beer foam formation (Lu et al. 2012). Beer LTP1 is degraded
13
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Page 8 of 12
by the released PrA; therefore, PrA directly affects the beer
foam stability (Zhang et  al. 2011). Meussdoerffer et  al.
(1980) reported that PrA is stable at temperatures lower than
45 °C and pH 5.5 and at 25 °C and pH of 5–6, indicating the
thermal stability of PrA. PrA denaturation is difficult due to
the PrA feature. Thus, the foam stability is decreased during
the storage of draft beer, which results from the stability of
PrA. A large amount of PrA is excreted in the yeasts with
low vitality; high yeast vitality should be selected in win-
emaking processes (Cooper et al. 2000).
Strategies to control PrA excretion in the beer
PrA is directed to the vacuole via the Golgi-to-vacuole route
under normal condition, and the CGPMP exports PrA from
the cells. Excreted PrA exerts an adverse influence on alco-
holic beverages, especially on the beer foam stability. There-
fore, PrA excretion should be controlled in beer. According
to previous studies, the current two methods for decreasing
PrA excretion are as follows: (1) fermentation process opti-
mization, including optimization of the concentrations of
nutritional elements in the fermentation broth, yeast separa-
tion in time from the fermentation broth, and addition of pro-
teinase inhibitors into the finished wine; (2) low PrA activity
strain construction, which is mainly obtained by means of
self-cloning technique.
Hao et al. (2010) reported that optimizing the content of
nutritional elements, including α-amino nitrogen, inorganic
salt, and biotin, could remarkably decrease PrA excretion.
The release of PrA from cells is evidently implicated in the
nutritional element concentrations of fermentation broth.
According to Kondo et al. (1999), optimized yeast cropping
timing leads to production of an ideal beer with low PrA
activity. The specific reason is that yeast autolysis results in
the increased extracellular PrA content at the end of fermen-
tation. Therefore, the yeast cells separated from the fermen-
tation broth in time decrease the PrA activity in the wort.
This finding is consistent with that of Lu et al. (2012). Nev-
ertheless, a small quantity of PrA is inevitably released into
the broth due to stress conditions in the fermentation pro-
cess. The commonly used method to lower the extracellular
PrA content is the addition of proteinase inhibitors, which
improve the stability of beer foam. PrA inhibitor pepstatin
A or proteinase inhibitor GLPAI1, which is separated from
Ganoderma lucidum, is added to the unpasteurized beer,
which prevents the decrease of beer foam stability (Tian and
Zhang 2005; Yokoi et al. 1996). Moreover, the addition of
soybean to beer can enhance the stability of beer foam due
to the protection of foam-active proteins from degradation
(Tian and Zhang 2005).
The most logical approach to improve foam stability via
genetic engineering is the construction of a PEP4 disruption
13
World J Microbiol Biotechnol (2017) 33:210
strain (Hao et al. 2008; Zhang et al. 2011). When a sin-
gle PEP4 in industrial strain is replaced with the bglS gene
(encoding endo-l,3-1,4-β-glucanase from Bacillus subti-
lis) expression cassette or the GSH1 gene (responsible for
γ-glutamylcysteine synthetase) expression cassette, no PrA
activity is measured in the fermenting liquor (Wang et al.
2007a, b; Zhang et al. 2008). Hao et al. (2008) reported that
allelic PEP4 genes are consecutively disrupted in Saccha-
romyces carlsbergensis, and a single PEP4 deletion mutant
significantly lowers foam stability. Nonetheless, no survival
mutants are ultimately gained when the second allelic PEP4
gene is knocked out.
The extracellular PrA activities of many-allele PEP4
gene disruption strains are lower than that of one-allele
PEP4 gene disruption strains, but the evident decrease in
wort sugar consumption and a long lag phase are observed
(Lu et al. 2012). Previous studies concluded that PEP4 gene
disruption contributes to the improvement of foam stabil-
ity. Furthermore, many allele PEP4 gene deletions in poly-
ploid industrial yeasts will considerably influence the cell
viability and fermentation performance of yeast. To solve
the growth defects of the above mutant, the excretion of
PrA should be suppressed under the premise of the unmodi-
fied PEP4 gene. PrA is missorted to the cell surface in the
cells lacking Vps10p; the missorting of proteinase A is sup-
pressed with overproduction of Vps10p (Cooper and Stevens
1996; Westphal et al. 1996). The PrA activity in the broth
fermented with Vps10p-overexpressing strain decreases to
23.04% of the host strain, and the fermentation performance
of the mutant is significantly improved (Chen et al. 2017).
The extracellular PrA activity in the SEC5 deletion strain
decreases to 77.61% of the host strain, and the fermenta-
tion performance between the mutant and the control strain
shows no evident differences (Chen et al. 2017). Evidently,
an ideal method for reduction of PrA excretion in S. cerevi-
siae is strengthening its Golgi-to-vacuole sorting or weak-
ening its constitutive Golgi-to-plasma membrane secretion,
which retains the PrA in the cells.
Prospect
The release of PrA into the broth affects the safety and qual-
ity of fermented products. The most common method of
decreasing PrA excretion is disruption of PEP4, and many
allelic PEP4 adversely influences the fermentation perfor-
mance of the industrial strains (Lu et al. 2012). Several stud-
ies indicated that strengthening the sorting of PrA to the
vacuole and preventing the release of PrA from the cells
can decrease PrA excretion, and they exert no significant
effect on fermentation performance (Chen et  al. 2017;
Westphal et al. 1996). These methods are two promising
methods for the reduction of PrA excretion. However, the
World J Microbiol Biotechnol (2017) 33:210 Page 9 of 12  210
current understanding of the precise mechanism of secre-
tory pathways of PrA is not complete. PrA propeptide con-
tains vacuolar sorting signal, and proPrA will be completely
degraded in the lack of prosequence (van den Hazel et al.
1993). Nevertheless, the degradation mechanism of proPrA
and the regulation mechanism of catalytic system of vacu-
olar enzymes related to PrA in the absence of proPrA remain
unclear. Previous findings indicated that four VSRs (Vps10p,
Mrl1p, Vth1p, and Vth2p) guide the PrA to the vacuole.
Hence, whether the additional sorting receptors exist in the
yeasts become a research object. In addition to Sec5p, spe-
cific components of the CGPMP are poorly understood. PrA
excretion may be a calcium-dependent process, but the pre-
cise mechanism regarding calcium ion for the regulation of
PrA excretion is still unknown. Calcium homeostasis in the
ER and the Golgi is implicated in PrA excretion. In future
research, we can control the excretion of PrA by disrupt-
ing calcium homeostasis and constructing low-PrA-activity
strains in the same manner. Future studies should be directed
at addressing the remaining remarkable questions. If the
existing questions are solved, then the understanding of the
mechanism of PrA excretion will be further complete. The
amino acid level in the fermentation broth will be precisely
regulated, which will significantly affect the nutrition, safety,
and sensory quality of alcoholic beverages associated with
amino acid content.
Acknowledgements  The current study was financially supported by
the program for the National Natural Science Foundation of China
(No. 31271916).
Compliance with ethical standards  PEP4 deletion in industrial S. cerevisiae wz65 on key enzymes
Conflict of interest  The authors declare that they have no conflict
of interest.
Ethical approval  For this type of study formal consent is not
required.
Informed consent  Informed consent was obtained from all indi-
vidual participants included in the study.
References
Abeijon C, Orlean P, Robbins PW et al (1989) Topography of gly-
cosylation in yeast: characterization of GDPmannose transport
and lumenal guanosine diphosphatase activities in Golgi-like
vesicles. Proc Nat Acad Sci USA 86:6935–6939. doi:10.1073/
pnas.86.18.6935
Aguilar CF, Cronin NB, Badasso M et al (1997) The three-dimensional
structure at 2.4 Å resolution of glycosylated proteinase a from
the lysosome-like vacuole of Saccharomyces cerevisiae. J Mol
Biol 267:899–915. doi:10.1006/jmbi.1996.0880
Ammerer G, Hunter CP, Rothman JH et al (1986) PEP4 gene of Sac-
charomyces cerevisiae encodes proteinase A, a vacuolar enzyme
required for processing of vacuolar precursors. Mol Cell Biol
6:2490–2499. doi:10.1128/MCB.6.7.2490
Antebi A, Fink GR (1992) The yeast Ca(2+)-ATPase homologue,
PMR1, is required for normal Golgi function and localizes in a
novel Golgi-like distribution. Mol Biol Cell 3:633. doi:10.1091/
mbc.3.6.633
Baranski TJ, Faust PL, Kornfeld S (1990) Generation of a lysosomal
enzyme targeting signal in the secretory protein pepsinogen. Cell
63:281–129. doi:10.1016/0092-8674(90)90161-7
Barlowe CK, Miller EA (2013) Secretory protein biogenesis and
traffic in the early secretory pathway. Genetics 193:383–410.
doi:10.1534/genetics.112.142810
Benli M, Döring F, Robinson DG et al (1996) Two GTPase isoforms,
Ypt31p and Ypt32p, are essential for Golgi function in yeast.
EMBO J 15:6460–6475
Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and
fusion. Cell 116:153–166. doi:10.1016/S0092-8674(03)01079-1
Booth C, Koch GL (1989) Perturbation of cellular calcium induces
secretion of luminal ER proteins. Cell 59(4):729–737.
doi:10.1016/0092-8674(89)90019-6
Brion C, Miller SG, Moore HP (1992) Regulated and constitutive
secretion. Differential effects of protein synthesis arrest on trans-
port of glycosaminoglycan chains to the two secretory pathways.
J Biol Chem 267:1477–1483
Bröcker C, Engelbrecht-Vandré S, Ungermann C (2010) Multisubunit
tethering complexes and their role in membrane fusion. Curr Biol
Cb 20:943–952. doi:10.1016/j.cub.2010.09.015
Bruno SNF, Vaitsman DS, Kunigami CN et al (2007) Influence of the
distillation processes from rio de janeiro in the ethyl carbamate
formation in brazilian sugar cane spirits. Food Chem 104:1345–
1352. doi:10.1016/j.foodchem.2007.01.048
Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of
proteins. Ann Rev Cell Biol 3:243–293. doi:10.1146/annurev.
cb.03.110187.001331
Chen S, Xu Y (2010) The influence of yeast strains on the volatile fla-
vour compounds of chinese rice wine. J Inst Brew 116:190–196.
doi:10.1002/j.2050-0416.2010.tb00417.x
Chen QH, Liu XJ, Fu ML et al (2010) Effect of PrA encoding gene-
in relation to the glycolytic pathway. Eur Food Res Technol
231:943–950. doi:10.1007/s00217-010-1355-y
Chen Y, Song L, Han Y et al (2017) Decreased proteinase a excretion
by strengthening its vacuolar sorting and weakening its consti-
tutive secretion in Saccharomyces cerevisiae. J Ind Microbiol
Biotechnol 44:149–159. doi:10.1007/s10295-016-1868-x
Cooper AA, Stevens TH (1996) Vps10p cycles between the late-
golgi and prevacuolar compartments in its function as the sort-
ing receptor for multiple yeast vacuolar hydrolases. J Cell Biol
133:529–541. doi:10.1083/jcb.133.3.529
Cooper DJ, Stewart GG, Bryce JH (2000) Yeast proteolytic activ-
ity during high and low gravity wort fermentations and its
effect on head retention. J Inst Brewtitute Brew 106:197–201.
doi:10.1002/j.2050-0416.2000.tb00057.x
Curwin AJ, von Blume J, Malhotra V (2012) Cofilin-mediated sorting
and export of specific cargo from the Golgi apparatus in yeast.
Mol Biol Cell 23:2327–2338. doi:10.1091/mbc.E11-09-0826
Dreyer T, Biedermann K, Ottesen M (1983) Yeast proteinase in beer.
Carlsberg Res Commun 48:249. doi:10.1007/BF02907771
Dreyer T, Halkier B, Svendsen I et al (1986) Primary structure of the
aspartic proteinase a from Saccharomyces cerevisiae. Carlsberg
Res Commun 51:27–41. doi:10.1007/BF02907993
Dürr G, Strayle J, Plemper R et al (1998) The medial-Golgi ion pump
Pmr1 supplies the yeast secretory pathway with ­Ca2+ and ­Mn2+
required for glycosylation, sorting, and endoplasmic reticulum-
associated protein degradation. Mol Biol Cell 9:1149–1162.
doi:10.1091/mbc.9.5.1149
13
210 
Page 10 of 12
Fokina AV, Chechenova MB, Karginov AV et al (2015) Genetic evi-
dence for the role of the vacuole in supplying secretory organelles
with ­Ca2+ in Hansenula polymorpha. PLoS ONE 10:e0145915.
doi:10.1371/journal.pone.0145915
Fuller RS, Brake A, Thorner J (1989) Yeast prohormone process-
ing enzyme (KEX2 gene product) is a C ­ a2+-dependent ser-
ine protease. Proc Nat Acad Sci USA 86:1434. doi:10.1073/
pnas.86.5.1434
Gibson BR, Lawrence SJ, Leclaire JP et  al (2007) Yeast
responses to stresses associated with industrial brew-
er y handling. FEMS Microbiol Rev 31:535–569.
doi:10.1111/j.1574-6976.2007.00076.x
Guo Y, Yang Y, Peng Q et al (2015a) Biogenic amines in wine: a
review. Int J Food Sci Technol 50:1523–1532. doi:10.1111/
ijfs.12833
Guo X, Guan X, Wang Y et al (2015b) Reduction of biogenic amines
production by eliminating the PEP4 gene in Saccharomyces cer-
evisiae during fermentation of Chinese rice wine. Food Chem
178:208. doi:10.1016/j.foodchem.2015.01.089
Gustchina A, Li M, Phylip LH et al (2002) An unusual orientation
for Tyr75 in the active site of the aspartic proteinase from
Saccharomyces cerevisiae. Biochem Biophys Res Commun
295:1020–1026
Hansen RJ, Switzer RL, Hinze H et al (1977) Effects of glucose and
nitrogen source on the levels of proteinases, peptidases, and pro-
teinase inhibitors in yeast. Biochim Biophys Acta 496:103–114.
doi:10.1016/0304-4165(77)90119-2
Hao J, Dong J, Speers R et al (2008) Construction of a single PEP4
allele deletion in saccharomyces carlsbergensis and a pre-
liminary evaluation of its brewing performance. J Inst Brew
114:322–328. doi:10.1002/j.2050-0416.2008.tb00776.x
Hao X, Xiao DG, Zhang CY et al (2010) Influence of nutrients on pro-
teinase a activity in draft beer during fermentation. Int J Food Sci
Technol 45:1169–1174. doi:10.1111/j.1365-2621.2010.02252.x
Harris SD, Cotter DA (1987) Vacuolar (lysosomal) trehalase of Saccha-
romyces cerevisiae. Curr Microbiol 15:247–249. doi:10.1007/
BF01589375
Hata T, Hayashi R, Doi E (1967) Purification of yeast proteinases:
part III. Isolation and physicochemical properties of yeast pro-
teinase A and ­C*. Agr Biol Chern 31:357–367. doi:10.1271/
bbb1961.31.150
Hirsch HH, Schiffer HH, Wolf DH (1992) Biogenesis of the yeast
vacuole (lysosome). Proteinase yscB contributes molecularly
and kinetically to vacuolar hydrolase-precursor maturation. Eur
J Biochem 207:867. doi:10.1111/j.1432-1033.1992.tb17118.x
Hong W, Lev S (2014) Tethering the assembly of SNARE complexes.
Trends Cell Biol 24:35–43. doi:10.1016/j.tcb.2013.09.006
Jones EW (1984) The synthesis and function of proteases in sac-
charomyces: genetic approaches. Ann Rev Gene 18:233–270.
doi:10.1146/annurev.ge.18.120184.001313
Jørgensen MU, Emr SD, Winther JR (1999) Ligand recognition and
domain structure of Vps10p, a vacuolar protein sorting recep-
tor in Saccharomyces cerevisiae. Eur J Biochem 260:461–469.
doi:10.1046/j.1432-1327.1999.00176.x
Kelly RB (1985) Pathways of protein secretion in eukaryotes. Science
230:25–32. doi:10.1126/science.2994224
Kienzle C, Von Blume J (2014) Secretory cargo sorting at the trans-
Golgi network. Trends Cell Biol 24:584–593. doi:10.1016/j.
tcb.2014.04.007
Klionsky DJ, Banta LM, Emr SD (1988) Intracellular sorting and pro-
cessing of a yeast vacuolar hydrolase: proteinase a propeptide
contains vacuolar targeting information. Mol Cell Biol 8:2105–
2116. doi:10.1128/MCB.8.5.2105
Klionsky DJ, Cueva R, Yaver DS (1992) Aminopeptidase I of Sac-
charomyces cerevisiae is localized to the vacuole independent
13
World J Microbiol Biotechnol (2017) 33:210
of the secretory pathway. J Cell Biol 119:287–299. doi:10.1083/
jcb.119.2.287
Kondo H, Shibano Y, Amachi T et al (1998) Substrate specificities and
kinetic properties of proteinase a from the yeast Saccharomyces
cerevisiae and the development of a novel substrate. J Biochem
124:141–147. doi:10.1093/oxfordjournals.jbchem.a022072
Kondo H, Yomo H, Furukubo S et al (1999) Advanced method for
measuring proteinase a in beer and application to brewing. J Inst
Brew 105:293–300. doi:10.1002/j.2050-0416.1999.tb00523.x
Lachenmeier DW (2007) Consequences of IARC re-evaluation of alco-
holic beverage consumption and ethyl carbamate on food control.
Dtsch Lebensmitt Rundsch 103:307–311
Lee KG (2013) Analysis and risk assessment of ethyl carbamate in
various fermented foods. Eur Food Res Technol 236:891–898.
doi:10.1007/s00217-013-1953-6
Lee S, Yoo M, Shin D (2015) The identification and quantification
of biogenic amines in Korean turbid rice wine, Makgeolli by
HPLC with mass spectrometry detection. LWT–Food Sci Tech-
nol 62:350–356. doi:10.1016/j.lwt.2015.01.016
Lenney JF, Dalbec JM (1967) Purification and properties of two pro-
teinases from Saccharomyces cerevisiae. Arch Biochem Biophys
120:42–48. doi:10.1016/0003-9861(67)90595-4
Liu JL, Guo W (2012) The exocyst complex in exocytosis and
cell migration. Protoplasma 249:587–597. doi:10.1007/
s00709-011-0330-1
Lodish HF, Kong N (1990) Perturbation of cellular calcium blocks exit
of secretory proteins from the rough endoplasmic reticulum. J
Biol Chem 265(19):10893–10899
Lu J, Dong J, Wu D et al (2012) Construction of recombinant indus-
trial brewer’s yeast with lower diacetyl production and proteinase
A activity. Eur Food Res Technol 235:951–961. doi:10.1007/
s00217-012-1821-9
Lundblad RL, Stein WH (1969) On the reaction of diazoacetyl com-
pounds with pepsin. J Biol Chem 244:154–160
Maddox IS, Hough JS (1970) Proteolytic enzymes of Saccharomyces
carlsbergensis. Biochem J 117:843–852. doi:10.1042/bj1170843
Marcusson EG, Horazdovsky BF, Cereghino JL et  al (1994) The
sorting receptor for yeast vacuolar carboxypeptidase Y is
encoded by the gene. Curr Biol 77:579–586. doi:10.1016/
S0960-9822(00)00231-1
Marks VD, Ho Sui SJ, Erasmus D et al (2008) Dynamics of the yeast
transcriptome during wine fermentation reveals a novel fermenta-
tion stress response. FEMS Yeast Res 8:35–52
Mechler B, Wolf DH (1981) Analysis of proteinase A function in
yeast. Eur J Biochem 121:47–52. doi:10.1111/j.1432-1033.1981.
tb06427.x
Meussdoerffer F, Tortora P, Holzer H (1980) Purification and proper-
tiesof proteinase a from yeast. J Biol Chem 255:12087–12093
Oda K (1992) Calcium depletion blocks proteolytic cleavages of
plasma protein precursors which occur at the Golgi and/or trans-
Golgi network. Possible involvement of Ca(2+)-dependent Golgi
endoproteases. J Biol Chem 267(24):17465–17471
Ortiz D, Medkova M, Walchsolimena C et al (2002) Ypt32 recruits
the Sec4p guanine nucleotide exchange factor, Sec2p, to secre-
tory vesicles; evidence for a Rab cascade in yeast. J Cell Biol
157:1005. doi:10.1083/jcb.200201003
Parr CL, Keates RAB, Bryksa BC et al (2007) The structure and func-
tion of Saccharomyces cerevisiae proteinase A. Yeast 24:467–
480. doi:10.1002/yea.1485
Peña-Gallego A, Hernández-Orte P, Cacho J et al (2012) High-per-
formance liquid chromatography analysis of amines in must and
wine: a review. Food Rev Int 28:71–96. doi:10.1080/87559129
.2011.594973
Pretorius IS (2000) Tailoring wine yeast for the new millennium:
novel approaches to the ancient art of winemaking. Yeast
World J Microbiol Biotechnol (2017) 33:210 Page 11 of 12  210
16:675–729. doi:10.1002/1097-0061(20000615)16:8<675::AID-
YEA585>3.3.CO;2-2
Pryer NK, Wuestehube LJ, Schekman R (1992) Vesicle-mediated
protein sorting. Annu Rev Biochem 61:471–516. doi:10.1146/
annurev.bi.61.070192.002351
Riffkin HL, Wilson R, Howie D et al (1989) Ethyl carbamate forma-
tion in the production of pot still whisky. J Inst Brewtitute Brew
95:115–119. doi:10.1002/j.2050-0416.1989.tb04618.x
Rothman JH, Hunter CP, Valls LA et al (1986) Overproduction-induced
mislocalization of a yeast vacuolar protein allows isolation of
its structural gene. Proc Nati Acad Sci USA 83:3248–3252.
doi:10.1073/pnas.83.10.3248
Rudolph HK, Antebi A, Fink GR et  al (1989) The yeast secre-
tory pathway is perturbed by mutations in PMR1, a
member of a ­ C a 2+ ATPase family. Cell 58:133–145.
doi:10.1016/0092-8674(89)90410-8
Saheki T, Holzer H (1975) Proteolytic activities in yeast. Biochim
Biophys Acta 384:203–214. doi:10.1016/0005-2744(75)90109-6
Saheki T, Matsuda Y, Holzer H (1974) Purification and characteriza-
tion of macromolecular inhibitors of proteinase A from yeast.
Eur J Biochem 47:325–332. doi:10.1111/j.1432-1033.1974.
tb03697.x
Sambrook JF (1990) The involvement of calcium in transport of secre-
tory proteins from the endoplasmic reticulum. Cell 61:197–199.
doi:10.1016/0092-8674(90)90798-J
Sanchez Y, Taulien J, Borkovich KA et al (1992) Hspl04 is required
for tolerance to many forms of stress. EMBO J 11:2357–2364.
doi:10.1111/j.1567-1364.2006.00035.x
Schehl B, Senn T, Lachenmeier DW et  al (2007) Contribution of
the fermenting yeast strain to ethyl carbamate generation in
stone fruit spirits. Appl Microbiol Biotechnol 74:843–850.
doi:10.1007/s00253-006-0736-4
Smit AY, Toit WJD, Toit MD (2008) Biogenic amines in wine: under-
standing the headache. South Afr J Enol Vitic 29:109–127.
doi:10.21548/29-2-1444
Söllner T, Whiteheart SW, Brunner M et al (1993) SNAP receptors
implicated in vesicle targeting and fusion. Nature 362:318–324.
doi:10.1038/362318a0
Somlyo AP, Bond M, Somlyo AV (1985) Calcium content of mitochon-
dria and endoplasmic reticulum in liver frozen rapidly in vivo.
Nature 314:622–625. doi:10.1038/314622a0
Springer S, Spang A, Schekman R (1999) A primer on vesicle budding.
Cell 97:145–148. doi:10.1016/S0092-8674(00)80722-9
Stevens TH, Rothman JH, Payne GS et al (1986) Gene dosage-depend-
ent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol
102:1551–1557. doi:10.1083/jcb.102.5.1551
Streb H, Bayerdörffer E, Haase W et al (1984) Effect of inositol-1,4,5-
trisphosphate on isolated subcellular fractions of rat pancreas. J
Membr Biol 81:241. doi:10.1007/BF01868717
Tang J (1971) Specific and irreversible inactivation of pepsin by sub-
strate-like epoxides. J Biol Chem 246:4510–4517
Teichert U, Mechlere B, Müller H, Wolf DH (1989) Lysosomal
(vacuolar) proteinases of yeast are essential catalysts for pro-
tein degradation, differentiation, and cell survival. J Biol Chem
264:16037–16045
Terbush DR, Novick P (1995) Sec6, Sec8, and Secl5 are components of
a multisubunit complex which localizes to small bud tips in Sac-
charomyces cerevisiae. J Cell Biol 130:299–312. doi:10.1083/
jcb.130.2.299
Tian YP, Zhang KC (2005) Purification and characterization of a
novel proteinase a inhibitor from Ganoderma lucidum by sub-
merged fermentation. Enzyme Microb Technol 36:357–361.
doi:10.1016/j.enzmictec.2004.10.003
Traub LM, Kornfeld S (1997) The trans-Golgi network: a late secre-
tory sorting station. Curr Opin Cel Biol 9:527–533. doi:10.1016/
S0955-0674(97)80029-4
Valls LA, Hunter CP, Rothman JH et al (1987) Protein sorting in
yeast: the localization determinant of yeast vacuolar car-
boxypeptidase Y resides in the propeptide. Cell 48:887–897.
doi:10.1016/0092-8674(87)90085-7
van den Hazel HB, Kielland-brandt MC, Winther JR (1992)
Autoactivation of proteinase A initiates activation of
yeast vacuolar zymogens. Eur J Biochem 207:277–283.
doi:10.1111/j.1432-1033.1992.tb17048.x
van den Hazel HB, Kielland-Brandt MC, Winther JR (1993) The
propeptideis required for in vivo formation of stable active
yeast proteinase a and can function even when not covalently
linked to the mature region. J Biol Chem 263:18002–18007
van den Hazel HB, Kielland-brandt MC, Winther JR (1995) Random
substitution of large parts of the propeptide of yeast proteinase
A. J Biol Chem 270:8602. doi:10.1074/jbc.270.15.8602
van den Hazel HB, Kielland-brandt MC, Wint her JR
(1996) Review: biosynthesis and function of yeast
va c u o l a r p rot e a s e s . Ye a st 1 2 : 1 – 1 6 . d o i : 1 0 . 1 0 0 2 /
(SICI)1097-0061(199601)12:1<1:AID-YEA902>3.0.CO;2-N
Von Blume J, Duran JM, Forlanelli E et al (2009) Actin remod-
eling by ADF/cofilin is required for cargo sorting at the trans-
Golgi network. J Cell Biol 187:1055–1069. doi:10.1083/
jcb.200908040
Von Blume J, Alleaume AM, Cantero-Recasens G et al (2011) ADF/
cofilin regulates secretory cargo sorting at the TGN via the
­Ca2+ ATPase SPCA1. Dev Cell 20:652–662. doi:10.1016/j.
devcel.2011.03.014
Wang ZY, He XP, Zhang BR (2007a) Over expression of GSH1 gene
and disruption of PEP4 gene in self-cloning industrial brew-
er’s yeast. Int J Food Microbiol 119:192–199. doi:10.1016/j.
ijfoodmicro.2007.07.015
Wang ZY, He GQ, Ruan H et al (2007b) Construction of proteinase A
deficient transformant of industrial brewing yeast. Eur Food Res
Technol 225:831–835. doi:10.1007/s00217-006-0488-5
Waters EJ, Alexander G, Muhlack R et al (2010) Preventing protein
haze in bottled white wine. Aust J Grape Wine Res 11:215–225.
doi:10.1111/j.1755-0238.2005.tb00289.x
Weber JV, Sharypov VI (2009) Ethyl carbamate in foods and bever-
ages: a review. Environ Chem Lett 7:233–247. doi:10.1007/
s10311-008-0168-8
Westphal V, Marcusson EG, Winther JR et al (1996) Multiple path-
ways for vacuolar sorting of yeast proteinase A. J Biol Chem
271:11865–11870
Whyte JRC, Munro S (2001) A yeast homolog of the mammalian
mannose 6-phosphate receptors contributes to the sorting of
vacuolar hydrolases. Curr Biol 11:1074–1078. doi:10.1016/
S0960-9822(01)00273-1
Wileman T, Kane LP, Carson GR, Terhorst C (1991) Depletion of cel-
lular calcium accelerates protein degradation in the endoplasmic
reticulum. J Biol Chem 266:4500
Wolff AM, Din N, Petersen JGL (1996) Vacuolar and
ex t r a c e l l u l a r m a t u r a t i o n o f S a c ch a ro m y c e s c e r-
evisiae proteinase A. Yeast 12:823. doi:10.1002/
(SICI)1097-0061(199607)12:9<823::AID-YEA975>3.0.CO;2-J
Woolford CA, Daniels LB, Park FJ et al (1996) The PEP4 gene encodes
an aspartyl protease implicated in the posttranslational regulation
of Saccharomyces cerevisiae vacuolar hydrolases. Mol Cell Biol
6:2500–2510. doi:10.1128/MCB.6.7.2500
Wu D, Chen Y, Li C et al (2016) Construction of self-cloning industrial
brewer’s yeast with SOD1 gene insertion into PEP4 prosequence
locus by homologous recombination. J Inst Brew 122:322–332.
doi:10.1002/jib.314
Yokoi S, Shigyo T, Tamaki T (1996) A fluorometric assay for pro-
teinase a in beer and its application for the investigation of
enzymatic effects on foam stability. J Inst Brew 102:33–37.
doi:10.1002/j.2050-0416.1996.tb00892.x
13
210 
Page 12 of 12
Zhang Q, Chen QH, Fu ML et al (2008) Construction of recombinant
industrial Saccharomyces cerevisiae strain with bglS gene inser-
tion into PEP4 locus by homologous recombination. J Zhejiang
Univ Sci B 9:527–535. doi:10.1631/jzus.B0820019
Zhang HB, Ruan H, Li WF et al (2011) Construction of recombinant
industrial S. cerevisiae strain with barley lipid-transfer protein 1
secretion capability and lower PrA activity. Eur Food Res Tech-
nol 233:707–716. doi:10.1007/s00217-011-1559-9
Zhao X, Du G, Zou H et al (2013) Progress in preventing the accumula-
tion of ethyl carbamate in alcoholic beverages. Trends Food Sci
Technol 32:97–107. doi:10.1016/j.tifs.2013.05.009
13
World J Microbiol Biotechnol (2017) 33:210
Zhao XR, Zou HJ, Fu JW et al (2014) Metabolic engineering of the
regulators in nitrogen catabolite repression to reduce the pro-
duction of ethyl carbamate in a model rice wine system. Appl
Environ Microbiol 80:392–398. doi:10.1128/AEM.03055-13
Zhong J, Ye X, Fang Z et  al (2012) Determination of biogenic
amines in semi-dry and semi-sweet Chinese rice wines from
the Shaoxing region. Food Control 28:151–156. doi:10.1016/j.
foodcont.2012.05.011
Zubenko GS, Park FJ, Jones EW (1982) Genetic properties of muta-
tions at the PEP4 locus in Saccharomyces cerevisiae. Genetics
102:679–690