Developmental Cell, Volume 37
Supplemental Information
Reticulons Regulate the ER Inheritance
Block during ER Stress
Francisco Javier Piña, Tinya Fleming, Kit Pogliano, and Maho Niwa
Inventory of Supplemental Materials
Figure S1, related to Figure 1. Contains representative images for class the I, II, III cER
inheritance phenotype (A) and representative images of ire1Δ (B) and slt2Δ (C) cells for
the FRAP experiments.
Figure S2, related to Figure 1. Contains graphs for the kar2-1-sfGFP FRAP experiments
(A and B) and representative images of cells for the Hmg1-GFP FRAP experiments (C).
Figure S3, related to Figure 4. Displays representative images for the ER phenotype of
WT, Δtether and rtn1Δrtn2Δyop1Δ cells (A), representative images of cells for FRAP
experiments with Δtether (B), rtn1Δrtn2Δyop1Δ (C), and rtn1Δrtn2Δyop1Δlnp1Δ (D)
cells, and images of tubule formation in WT, slt2Δ, rtn1Δrtn2Δyop1Δ, and
rtn1Δrtn2Δyop1Δlnp1Δ cells (E).
Figure S4, related to Figure 4. FRAP analysis for Hmg1-GFP in rtn1Δrtn2Δyop1Δ cells
(A). Shows a cartoon model depicting ER stress, tubule formation, and cER inheritance
in WT cells vs different mutant strains used in the study (B and C).
Supplemental Experimental Procedures
ER inheritance assay
CPY*-mRFP and GFP-CFTR assays
Construction of Kar2-sfGFP strains
Yeast strains used
Plasmids used
Primers used
Supplemental References
Supplemental Figure 1
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Supplemental Figure 1. ER stress causes a block in inheritance of cER but not
pnER. Related to Figure 1.
(A) ER inheritance was monitored in Hmg1-GFP-expressing WT cells treated with
DMSO (-Tm) or 1 µg/ml Tm for 3 hr. Representative cells are shown for class I, II and III
with small, medium, and large buds, respectively.
(B–C) Representative images of ire1Δ (B) and slt2Δ (C) cells from Kar2-sfGFP FRAP
experiments. Cells were incubated with DMSO or 1 µg/ml Tm for 3 hr before small
regions of the cER (orange box) or pnER (green box) were photobleached. Images were
acquired before (pre-bleach), at the same time as (bleach), and at 18 or 84 sec after
photobleaching. Scale bar is 2 µm.
Supplemental Figure 2. FRAP analysis of Hmg1-GFP-expressing WT cells and
mutant kar2-1-sfGFP-expressing cells. Related to Figure 1.
(A and B) kar2-1-sfGFP cells (Kabani et al., 2003) were incubated with DMSO or 1 µg/ml
Tm for 30 min or 3 hr before FRAP in the cER (B) and pnER (C) were analyzed. Graphs
are the mean ± SD of 3 experiments, each examining ≥7 cells. Scale bar is 2 µm.
(C) WT cells expressing Hmg1-GFP were incubated with DMSO or 1 µg/ml Tm before a
small region of the cER (orange box) or pnER (green box) was photobleached. Images
were acquired before (pre-bleach), at the same time as (bleach), and at 18 or 84 sec
after photobleaching. Scale bar is 2 µm.
Supplemental Figure 3. FRAP analysis of Kar2-sfGFP-expressing Δtether,
rtn1Δrtn2Δyop1Δ , and rtn1Δrtn2Δyop1Δ lnp1Δ cells. Related to Figure 3 and 4.
(A) Δtether and rtn1Δrtn2Δyop1Δ cells have defective ER morphology and distribution. In
WT cells, the ER is juxtaposed to the PM and appears as a continuous ring around the
periphery of the cell and the nucleus. In Δtether cells, the cER is discontinuous and is
not in close apposition to the PM. In rtn1Δrtn2Δyop1Δ cells, the cER appears to be
discontinuous around the periphery of the cell with invaginations displaced from the PM.
Scale bar is 2 µm.
(B-D) Representative images from FRAP experiments with Δtether (B), rtn1Δrtn2Δyop1Δ
(C), and rtn1Δrtn2Δyop1Δlnp1Δ (D) cells expressing Kar2-sfGFP. Cells were treated with
DMSO or 1 µg/ml Tm for 3 hr before FRAP in the pnER (green box) or cER (orange box)
was assessed. Scale bars are 2 µm.
(E) Images of tubule formation in WT, slt2Δ, rtn1Δrtn2Δyop1Δ, and
rtn1Δrtn2Δyop1Δlnp1Δ cells expressing Hmg1-GFP. Tubule formation from the pnER
was examined early in the cell cycle during bud initiation in cells incubated with DMSO
or 1 µg/ml Tm for 3 hr. ER stress reduces or causes abnormal ER tubule formation.
Scale bar is 2 µm.
Supplemental Figure 4. Model for Slt2 and RTN function in ER tubule formation. Related to Figure 4. (A) FRAP analysis in unstressed (DMSO, 3hr) or stressed (Tm, 3hr) cER or pnER of
slt2Δ cells expressing Hmg1-GFP. Graphs represent the mean ± SD of 3 experiments,
each examining ≥7 cells.
(B) FRAP analysis in unstressed (DMSO, 3hr) or stressed (Tm, 3hr) cER or pnER of
rtn1Δrtn2Δyop1Δ cells expressing Hmg1-GFP. Graphs represent the mean ± SD of 3
experiments, each examining ≥7 cells.
(C, left) Under normal growth conditions (-Tm), the cER and pnER have similar
environments with high mobility of the ER-resident chaperone Kar2/BiP reporter, Kar2-
sfGFP. At a specific but currently unknown point in the cell cycle, tubular ER emerges
from the pnER orientated towards the bud. We propose that a balance in the activity or
localization of ER shaping proteins (Rtn1/2, Yop1, Sey1, and Lnp1) at the three-way
junction formed between tubular ER at the pnER regulates tubule formation from the
pnER and orientation toward the new bud site. Ultimately, the tubular ER enters the
daughter cell, becomes anchored to the bud tip, and spreads around the cortex.
(C, right) When ER stress is induced (+Tm) in WT cells, Kar2-sfGFP mobility decreases
in the cER, but not the pnER. Thus, the combination of (1) an unperturbed pnER
environment, (2) a block in the emergence of tubular ER from the pnER, and (3) a
subsequent block in cER inheritance can be described as the “WT ER stress
phenotype.” This requires Slt2 activation, which may alter the balance between the
Reticulons, Yop1, Lnp1 and Sey1.
(D, left) Maintenance of an unperturbed pnER environment under conditions of ER
stress requires Slt2 activation. Thus, in slt2Δ cells, ER stress decreases Kar2-sfGFP
mobility to a similar extent in the cER and the pnER and both the block in tubular ER
emergence and the block in cER inheritance are abolished, possibly due to inability to
alter the balance of ER structural proteins. Note that the establishment of asymmetric
cER and pnER environments is not simply due to disturbance in ER architecture, as loss
of ER–PM contact sites in Δtether cells had no effect on the mobility of Kar2-sfGFP
under ER stress conditions. Rather, the pnER and cER environments appear to be
differentially affected by components affecting the local ER structure.
(D, middle) In rtn1Δrtn2Δyop1Δ cells, Kar2-sfGFP mobility decreases in both the cER
and pnER under conditions of ER stress, demonstrating that the RTNs/Yop1 are
necessary to maintain the unperturbed pnER environment. Moreover, rtn1Δrtn2Δyop1Δ
cells do not form tubular ER from the pnER, even in the absence of ER stress,
illustrating the importance of the RTNs/Yop1 for ER inheritance. Importantly,
rtn1Δrtn2Δyop1Δ cells are incapable of responding to ER stress even though Slt2 is
active.
(D, right) Deletion of LNP1 in rtn1Δrtn2Δyop1Δ cells re-establishes their ability to
respond to ER stress. The unperturbed pnER environment, block in the initial formation
of tubular ER, and the block in cER inheritance are all re-established, although the latter
two do not reach WT levels.
We propose that the ability to generate a stable tubular ER rests on the establishment of
ER membrane characteristics determined by a balance between the Reticulons, Yop1,
Lnp1 and Sey1. A previous study has demonstrated that Slt2 is transiently activated
during the normal cell cycle (Li et al., 2010), and we speculate that this may cause a
delay in initiating the formation of the tubular ER from the pnER. Such a transient block
might serve as a cell cycle checkpoint that provides a window of opportunity to activate
the ERSU pathway if the ER functional status is not conducive to proceeding with cell
division.
Supplemental Experimental Procedures
ER inheritance assays: Cells expressing Hmg1-GFP were treated with DMSO or Tm
(1 µg/ml) for 3 hr during mid-log phase, imaged with fluorescence microscopy, and
scored for the presence or absence of cER in class I, class II, and class III buds, as
described previously (Babour et al., 2010). Strains CVY25 (Anwar et al., 2012),
ANDY198 (Manford et al., 2012), MNY2850, MNY2851, and MNY2852 (Chen et al.,
2012) were transformed with pRH475 (digested with StuI) to generate strains expressing
Hmg1-GFP. An Axiovert 200M Carl Zeiss Micro-Imaging microscope with a 100× 1.3 NA
objective was used for imaging as described previously (Babour et al., 2010). The
graphs represent the mean ± SD of 3 experiments, each analyzing ≥200 cells for cER
inheritance.
CPY* and CFTR foci formation assays: MNY1037 was transformed with pFJP10 and
cells were grown overnight on synthetic media without Leucine with 4% raffinose. Cells
were diluted to OD 0.06 and grown to OD 0.25, at which point 2% dextrose (+ DMSO or
1 µg/mL Tm) or 2% galactose (+ DMSO or 1 µg/mL Tm) was added. Cultures were
further incubated at 30ºC for 2 hr before cells were visualized by fluorescence
microscopy to examine CPY*-mRFP foci formation. MNY2215 (DsRed-HDEL) was
transformed with pCU426CUP1/EGFP-CFTR. Cells were grown overnight in synthetic
media without uracil containing 2% dextrose, diluted to OD 0.06 and grown to OD 0.25,
at which point 100 µM copper sulfate plus DMSO or 1 µg/mL Tm was added. Cultures
were further incubated at 30ºC for 2 hr before cells were visualized by fluorescence
microscopy to examine GFP-CFTR foci formation. The graphs represent the mean ± SD
of 3 experiments, each analyzing ≥100 cells for aggregate formation.
Construction of KAR2-sfGFP strains: pFA6a-GFP::Kanr was modified by replacing
GFP with sfGFP-HDEL. sfGFP-HDEL was PCR amplified from plasmid psfGFPHDEL
with primers containing PacI and AscI restriction sites, and the products were digested
and ligated into PacI- and AscI-digested pFA6a-GFP::Kanr. The plasmid was checked by
sequencing. sfGFP-HDEL::Kanr was PCR amplified with primers FJP17 and FJP37 to
tag KAR2 at the genomic locus of the indicated strains using the Longtine method
(Longtine et al., 1998). The KanMX marker was swapped for URA3 with plasmid M4758
(Voth et al., 2003) (digested with NotI) in yeast strain MNY2119. SLT2 was knocked out
with PCR product from primers OFY80 and OFY81 in strain MNY2119 to generate
MNY2121. KAR2 was tagged with sfGFP-HDEL::URA3 at the genomic locus in strains
CVY25 (Anwar et al., 2012), ANDY198 (Manford et al., 2012), MNY2850, MNY2851, and
MNY2852 (Chen et al., 2012).
Yeast strains used in this study
Yeast Strain Genotype Reference MNY2119 MATa, leu2-3,112, trp1-1, can1-100, ura3-
1, ade2-1, his3-11,15::UPRE-lacZ:HIS3 KAR2-sfGFP::KanMX
(Pina and Niwa, 2015)
MNY2121 MATa, leu2-3,112, trp1-1, can1-100, ura3-1, ade2-1, his3-11,15::UPRE-lacZ::HIS3 KAR2sfGFP::KanMX slt2Δ::NatMX
This study
MNY2120 MATa, leu2-3,112, trp1-1, can1-100, ura3-1, ade2-1, his3-11,15::HIS3, bar1Δ::LEU2, ire1Δ::KanMX KAR2-sfGFP::NatMX
This study
MNY2703 MATa leu2-3,-112 his 3-11,-15 trp1-1 ura3-1::HMG1-GFP:URA3 ade2-1 ade3 rtn1Δ::kanMX4 rtn2Δ::kanMX4 yop1Δ::kanMX4
This study
MNY1037 MATa, leu2-3,112, trp1-1, can1-100, ura3-1::HMG1-GFP:URA3, ade2-1, his3-11,15::UPRE-lacZ:HIS3
(Babour et al., 2010)
MNY2038 Ire1-GFP strain (pWY1555) (Aragon et al., 2009)
MNY2705 MATa leu2-3,-112 his 3-11,-15 trp1-1 ura3-1 ade2-1 ade3 rtn1Δ::kanMX4 rtn2Δ::kanMX4 yop1Δ::kanMX4 KAR2sfGFP::URA3
This study
MNY2706 MATa leu2‐3,112 ura3‐52 his3‐Δ200 trp1‐Δ901 lys2‐801 suc2‐Δ9 ist2Δ::HISMX6 scs2Δ::TRP1 scs22Δ::HISMX6 tcb1Δ::KANMX6 tcb2Δ::KANMX6 tcb3Δ::HISMX6 KAR2-sfGFP::URA3
This study
MNY2707 MATa leu2‐3,112 ura3‐52::HMG1-GFP:URA3 his3‐Δ 200 trp1‐Δ901 lys2‐801 suc2‐Δ9 ist2Δ::HISMX6 scs2Δ::TRP1 scs22Δ::HISMX6 tcb1Δ::KANMX6 tcb2Δ::KANMX6 tcb3Δ::HISMX6
This study
MNY2704 MNY2038 DsRed-HDEL::ADE2 This study
MNY1043 MATa, leu2-3,112, trp1-1, can1-100, ura3-1::HMG1-GFP:URA3, ade2-1, his3-
(Babour et al., 2010)
11,15::UPRE-lacZ:HIS3, slt2Δ::KanMX MNY2845 MATa, ura3-52, leu2-3,112, his3Δ200,
lnp1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ:: NatMX, yop1Δ::NatMX, KAR-2sfGFP::URA3
This study
MNY2846 MATa, ura3-52, leu2-3,112, his3Δ200, lnp1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ::NatMX, yop1Δ::NatMX, HMG1-GFP::URA3
This study
MNY2355 MATa ura3-52, leu2-3,112 ade2-101 kar2-1 HIS3 kar2-1-sfGFP::KanMX
This study
MNY2848 MATa, ura3-52, leu2-3,112, his3Δ200, sey1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ:: NatMX, yop1Δ:: NatMX, KAR-2sfGFP::URA3
This study
MNY2849 MATa, ura3-52, leu2-3,112, his3Δ200, sey1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ::NatMX, yop1Δ::NatMX, HMG1-GFP::URA3
This study
MNY2850 MATa, ura3-52, leu2-3,112, his3Δ200, sey1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ::NatMX, yop1Δ::NatMX
(Chen et al., 2012)
CVY25 MATa leu2-3,-112 his 3-11,-15 trp1-1 ura3-1 ade2-1 ade3 rtn1Δ::kanMX4 rtn2Δ::kanMX4 yop1Δ::kanMX4
(Anwar et al., 2012)
ANDY198 MATa leu2‐3,112 ura3‐52 his3‐Δ 200 trp1‐Δ901 lys2‐801 suc2‐Δ9 ist2Δ::HISMX6 scs2Δ::TRP1 scs22Δ::HISMX6 tcb1Δ::KANMX6 tcb2Δ::KANMX6 tcb3Δ::HISMX6
(Manford et al., 2012)
MNY2851 MATa, ura3-52, leu2-3,112, his3Δ200, lnp1Δ::His3MX6, rtn1Δ::KanMX6, rtn2Δ::NatMX, yop1Δ::NatMX
(Chen et al., 2012)
MNY2852 MATa, ura3-52, leu2-3,112, his3Δ200 (Chen et al., 2012)
Plasmids used in this study
Plasmid name Construct Reference
pFJP10 pGAL1-CPY*-mRFP (Pina and Niwa, 2015) GFP-CFTR (pCU426CUP1/EGFP-CFTR) (Fu and Sztul, 2003) pFJP1 pFA6a-sfGFP-HDEL::Kanr (Pina and Niwa, 2015) pRH475 Hmg1-GFP::URA3 (Hampton et al., 1996)
Primers used in this study
Primer name Sequence Use
FJP17 ATAAATTAACAACCTTGAAGCTTCCAGCAGCAAAAATTTTTAACTATTTTATgaattcgagctcgtttaaac
Forward primer to tag KAR2 with sfGFP-HDEL::KanMX
FJP37 CAGTCTCTATACTCTTCAATG Reverse primer to tag KAR2 with sfGFP-HDEL::KanMX
OFY80 TAGAAATAATTGAAGGGCGTGTATAACAATTCTGGGAGGACGTCGCCTCGTCCCCGCCGGGTCACCC
Forward primer to knockout SLT2 and replace with NatMX
OFY81 GTGATTCTATACTTCCCCGGTTACTTATAGTTTTTTGTCCGACGTCAGCAGTATAGCGACCAGCATTCAC
Reverse primer to knockout SLT2 and replace with NatMX
FJP13 GGCttaattaacATGGTGAGCAAGGGCGAGGAG
Forward primer (PacI endonuclease site) to replace the GFP sequence in the pFA6a-GFP-Kanr plasmid
FJP14 GGggcgcgccTTACAATTCATCGTGGTACAG
Reverse primer (AscI endonuclease site) with HDEL to replace the GFP sequence in the pFA6a-GFP-Kanr plasmid
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