from imbalances between demand and capacity
on the protein folding machinery in the endoplasmic reticulum (ER). A consequence of such
imbalances that may arise from different intracellular and extracellular influences on ERlocalized or secreted proteins is the accumulation of unfolded or misfolded proteins in the
ER lumen. Detection of these potentially toxic
or harmful proteins (preproteins/protein intermediates) occurs by the ER membranelocalized sensor proteins PERK (Protein kinase
R (PKR)-like Endoplasmic Reticulum Kinase),
ATF6 (Activating Transcription Factor 6), and
Ire1 (Inositol requiring enzyme 1), of which
only the latter is conserved from fungi to mammals and best studied in the baker’s yeast Saccharomyces cerevisiae. Ire1 is composed of an
ER luminal sensor-domain that is connected by
an ER membrane-spanning transmembrane
domain to the cytoplasmic part harboring
kinase and RNase functions (Fig. 3.1), both of
which are required for the induction of ER
stress-responsive genes (Kohno et al. 1993;
Kozutsumi et al. 1988; Mori et al. 1993; Rose
et al. 1989).
In S. cerevisiae, sensing of unfolded or misfolded proteins in the ER lumen can occur by
(1) direct binding of misfolded proteins to the
ER luminal sensor domain or indirectly by (2)
unfolded protein-dependent depletion of the
major ER chaperone Kar2/Bip1 (Karyogamy/
Binding immunoglobulin protein) that under
unstressed conditions binds to the Ire1 sensor
domain and prevents formation of oligomeric
and active Ire1 complexes (Zhou et al. 2006). In
higher eukaryotes, activation of ATF6 and
PERK is similarly triggered by the reduced
interaction between Bip1 and ATF6 or PERK.
In case of ATF6 this leads to relocalization of
ATF6 to the Golgi and subsequent S2P (Site
2 Protease)-mediated cleavage to liberate the
transcriptionally active ATF6 bZIP protein
(Ron and Walter 2007). In case of PERK, activation of its kinase domain promotes phosphorylation of eIF2a (eukaryotic Initiation
Factor 2a) to globally inhibit protein biosynthesis via attenuated translation initiation. This
mode of action is analogous to GCN2 (General
Control Nonderepressible 2)-dependent phosphorylation of eIF2a under amino acid starvation
conditions
(Hinnebusch
1994).
Interestingly, all three pathways share common
principles in their mode of activation and individually activate expression of specific bZIP
transcription factors that mediate the transcriptional response towards ER stress.
B. Ire1 and Hac1: Key Players of the UPR
1. Activation of Ire1
In S. cerevisiae, detection of unfolded proteins
in the ER lumen triggers Ire1 oligomerisation,
trans-autophosphorylation, ADP binding, and
activation of the RNAse domain (Gardner et al.
2013) that resides on the cytoplasmic portion of
the Ire1 protein. Activity of Ire1 is induced in
response to a wide variety of different stimuli.
These affect either protein folding in the ER,
such as increased demands for protein secretion, or treatment with protein folding inhibitors (dithiothreitol, DTT; tunicamycin, TM)
and heat stress. In addition, Ire1 is also activated by ER membrane aberrancies associated
with or connected to cell wall stress, inositol
depletion (Gardner et al. 2013; Gardner and
Walter 2011; Korennykh et al. 2011; Rubio
et al. 2011), and iron depletion (Cohen et al.
2017). A multitude of different compounds has
been identified that either directly or indirectly
activate Ire1 and induce the UPR, for most of
which the mode of action has not been fully
resolved. Interestingly, quercetin, a plantderived flavanone, directly binds to an unconventional ligand binding pocket and promotes
increased Ire1 activity in vitro (Wiseman et al.
2010). In Aspergillus nidulans and Candida
albicans, the isoprenoid alcohol farnesol and
the monoterpene carvacrol trigger UPR activity, respectively (Chaillot et al. 2015; Colabardini et al. 2010). The activity of Ire1 is moreover
modulated by various factors. For example, in
S. cerevisiae, the phosphatase Ptc2 (Phosphatase Two C 2) mediates Ire1 dephosphorylation
and thus contributes to inactivation of the UPR
(Welihinda et al. 1998). Interestingly, Ire1
mutants lacking the trans-autophosphorylation
activity do not show loss of UPR activity, but
prolonged UPR activation, suggesting that
50
R. Harting and K. Heimel
on the protein folding machinery in the endoplasmic reticulum (ER). A consequence of such
imbalances that may arise from different intracellular and extracellular influences on ERlocalized or secreted proteins is the accumulation of unfolded or misfolded proteins in the
ER lumen. Detection of these potentially toxic
or harmful proteins (preproteins/protein intermediates) occurs by the ER membranelocalized sensor proteins PERK (Protein kinase
R (PKR)-like Endoplasmic Reticulum Kinase),
ATF6 (Activating Transcription Factor 6), and
Ire1 (Inositol requiring enzyme 1), of which
only the latter is conserved from fungi to mammals and best studied in the baker’s yeast Saccharomyces cerevisiae. Ire1 is composed of an
ER luminal sensor-domain that is connected by
an ER membrane-spanning transmembrane
domain to the cytoplasmic part harboring
kinase and RNase functions (Fig. 3.1), both of
which are required for the induction of ER
stress-responsive genes (Kohno et al. 1993;
Kozutsumi et al. 1988; Mori et al. 1993; Rose
et al. 1989).
In S. cerevisiae, sensing of unfolded or misfolded proteins in the ER lumen can occur by
(1) direct binding of misfolded proteins to the
ER luminal sensor domain or indirectly by (2)
unfolded protein-dependent depletion of the
major ER chaperone Kar2/Bip1 (Karyogamy/
Binding immunoglobulin protein) that under
unstressed conditions binds to the Ire1 sensor
domain and prevents formation of oligomeric
and active Ire1 complexes (Zhou et al. 2006). In
higher eukaryotes, activation of ATF6 and
PERK is similarly triggered by the reduced
interaction between Bip1 and ATF6 or PERK.
In case of ATF6 this leads to relocalization of
ATF6 to the Golgi and subsequent S2P (Site
2 Protease)-mediated cleavage to liberate the
transcriptionally active ATF6 bZIP protein
(Ron and Walter 2007). In case of PERK, activation of its kinase domain promotes phosphorylation of eIF2a (eukaryotic Initiation
Factor 2a) to globally inhibit protein biosynthesis via attenuated translation initiation. This
mode of action is analogous to GCN2 (General
Control Nonderepressible 2)-dependent phosphorylation of eIF2a under amino acid starvation
conditions
(Hinnebusch
1994).
Interestingly, all three pathways share common
principles in their mode of activation and individually activate expression of specific bZIP
transcription factors that mediate the transcriptional response towards ER stress.
B. Ire1 and Hac1: Key Players of the UPR
1. Activation of Ire1
In S. cerevisiae, detection of unfolded proteins
in the ER lumen triggers Ire1 oligomerisation,
trans-autophosphorylation, ADP binding, and
activation of the RNAse domain (Gardner et al.
2013) that resides on the cytoplasmic portion of
the Ire1 protein. Activity of Ire1 is induced in
response to a wide variety of different stimuli.
These affect either protein folding in the ER,
such as increased demands for protein secretion, or treatment with protein folding inhibitors (dithiothreitol, DTT; tunicamycin, TM)
and heat stress. In addition, Ire1 is also activated by ER membrane aberrancies associated
with or connected to cell wall stress, inositol
depletion (Gardner et al. 2013; Gardner and
Walter 2011; Korennykh et al. 2011; Rubio
et al. 2011), and iron depletion (Cohen et al.
2017). A multitude of different compounds has
been identified that either directly or indirectly
activate Ire1 and induce the UPR, for most of
which the mode of action has not been fully
resolved. Interestingly, quercetin, a plantderived flavanone, directly binds to an unconventional ligand binding pocket and promotes
increased Ire1 activity in vitro (Wiseman et al.
2010). In Aspergillus nidulans and Candida
albicans, the isoprenoid alcohol farnesol and
the monoterpene carvacrol trigger UPR activity, respectively (Chaillot et al. 2015; Colabardini et al. 2010). The activity of Ire1 is moreover
modulated by various factors. For example, in
S. cerevisiae, the phosphatase Ptc2 (Phosphatase Two C 2) mediates Ire1 dephosphorylation
and thus contributes to inactivation of the UPR
(Welihinda et al. 1998). Interestingly, Ire1
mutants lacking the trans-autophosphorylation
activity do not show loss of UPR activity, but
prolonged UPR activation, suggesting that
50
R. Harting and K. Heimel