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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
B slt2Δ Kar2sfGFP bleach 18 sec 84 sec
Tm D
M S
O Tm
D M
S O
pre-bleach ire1Δ Kar2sfGFP bleach 18 sec 84 sec
Tm D
M S
O Tm
D M
S O
pre-bleach
C
A No ER stress (-Tm) ER stress (+Tm)
m ed
iu m
la rg
e sm
al l
cE R
pn E
R
cE R
pn E
R
Supplemental Figure 2
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
kar2-1-sfGFP
kar2-1-sfGFP
3 hrs
3 hrs
yrevoce R ecnecseroul
F 0.0 0.2 0.4 0.6 0.8 1.0 1.2
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
kar2-1-sfGFP 30 min
A
B
1.2yrevoce R ecnecseroul
F 0.0 0.2 0.4 0.6
0.8
1.0
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
kar2-1-sfGFP 30 min
cER
pnER
C Wildtype Hmg1-GFP
pre-bleach bleach 18 sec 84 sec
Tm D
M S
O Tm
D M
S O
cE R
pn E
R
D M
S O
Tm A
B
Δtetherwildtype rtn1Δrtn2Δyop1Δ
pre-bleach bleach 18 sec 84 sec Δtether Kar2sfGFP
D M
S O
Tm
rtn1Δrtn2Δyop1Δlnp1Δ Kar2sfGFP pre-bleach bleach 18 sec 84 sec
D M
S O
Tm D
M S
O Tm
D
C rtn1Δrtn2Δyop1Δ Kar2sfGFP
pre-bleach bleach 18 sec 84 sec
D M
S O
Tm D
M S
O Tm
Supplemental Figure 3 D
M S
O Tm
slt2Δwildtype rtn1Δrtn2Δ yop1Δ
rtn1Δrtn2Δ yop1Δlnp1Δ
E
cE R
pn E
R
cE R
pn E
R cE
R pn
E R
Supplemental figure 4
D
slt2Δ
ER Inheritance no Slt2 Slt2 (active)
rtn1Δrtn2Δyop1Δ
Slt2 (active) no
ER Inheritance
rtn1Δrtn2Δyop1Δlnp1Δ +Tm
C +Tm
Slt2 (active) noER Inheritance
-Tm
ER Inheritance
A
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
rtn1Δrtn2Δyop1Δ
time (sec) -1 0 6 18 30 42 54 66 78
Tm
DMSO
rtn1Δrtn2Δyop1Δ
0.0 0.2 0.4
0.6 0.8 1.0 1.2yrevoce
R ecnecseroul F
Hmg1-GFPHmg1-GFP
cER pnER
0.0 0.2 0.4
0.6 0.8 1.0 1.2yrevoce
R ecnecseroul F
time (sec) -1 0 6 18 30 42 54 66 78
time (sec) -1 0 6 18 30 42 54 66 78
slt2Δ
cER pnER slt2Δ
B
Tm
DMSO
Tm
DMSOHmg1-GFPHmg1-GFP
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