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Page 1: Developmental Cell, Volume 37€¦ · Developmental Cell, Volume 37 Supplemental Information Reticulons Regulate the ER Inheritance Block during ER Stress ... 2010). Strains CVY25

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

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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

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Supplemental Figure 1

Bslt2Δ Kar2sfGFPbleach 18 sec 84 sec

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Supplemental Figure 2

time (sec)-1 0 6 18 30 42 54 66 78

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DM

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TmA

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Δtetherwildtype rtn1Δrtn2Δyop1Δ

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rtn1Δrtn2Δyop1Δlnp1Δ Kar2sfGFPpre-bleach bleach 18 sec 84 sec

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Supplemental Figure 3D

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Supplemental figure 4

D

slt2Δ

ER Inheritanceno Slt2 Slt2 (active)

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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.

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(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

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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.

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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

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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)

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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

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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

Supplemental References Anwar K, Klemm RW, Condon A, Severin KN, Zhang M, Ghirlando R, Hu J, Rapoport

TA, Prinz WA. 2012. The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae. The Journal of Cell Biology, 197, 209-217. doi: 10.1083/jcb.201111115

Aragon T, Van Anken E, Pincus D, Serafimova IM, Korennykh AV, Rubio CA, Walter P. 2009. Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature, 457, 736-40. doi: 10.1038/nature07641

Babour A, Bicknell AA, Tourtellotte J, Niwa M. 2010. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance. Cell, 142, 256-69. doi: 10.1016/j.cell.2010.06.006

Chen S, Novick P, Ferro-Novick S. 2012. ER network formation requires a balance of the dynamin-like GTPase Sey1p and the Lunapark family member Lnp1p. Nature cell biology, 14, 707-16. doi: 10.1038/ncb2523

Fu L, Sztul E. 2003. Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator. The Journal of Cell Biology, 160, 157-63. doi: 10.1083/jcb.200210086

Hampton RY, Koning A, Wright R, Rine J. 1996. In vivo examination of membrane protein localization and degradation with green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 93, 828-33. doi:

Kabani M, Kelley SS, Morrow MW, Montgomery DL, Sivendran R, Rose MD, Gierasch LM, Brodsky JL. 2003. Dependence of endoplasmic reticulum-associated

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Manford AG, Stefan CJ, Yuan HL, Macgurn JA, Emr SD. 2012. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Developmental cell, 23, 1129-40. doi: 10.1016/j.devcel.2012.11.004

Pina FJ, Niwa M. 2015. The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance. eLife, 4. doi: 10.7554/eLife.06970

Voth WP, Jiang YW, Stillman DJ. 2003. New 'marker swap' plasmids for converting selectable markers on budding yeast gene disruptions and plasmids. Yeast, 20, 985-93. doi: 10.1002/yea.1018