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• W2336735095 abstract "•The cortical ER and perinuclear (pn) ER respond to ER stress asymmetrically•Reduced asymmetry of the ER stress response blocks initial tubular ER formation•Initial tubule formation from the pnER is a key determinant of ER inheritance•Reticulons/Yop1 govern the asymmetric ER stress response and ER inheritance block Segregation of functional organelles during the cell cycle is crucial to generate healthy daughter cells. In Saccharomyces cerevisiae, ER stress causes an ER inheritance block to ensure cells inherit a functional ER. Here, we report that formation of tubular ER in the mother cell, the first step in ER inheritance, depends on functional symmetry between the cortical ER (cER) and perinuclear ER (pnER). ER stress induces functional asymmetry, blocking tubular ER formation and ER inheritance. Using fluorescence recovery after photobleaching, we show that the ER chaperone Kar2/BiP fused to GFP and an ER membrane reporter, Hmg1-GFP, behave differently in the cER and pnER. The functional asymmetry and tubular ER formation depend on Reticulons/Yop1, which maintain ER structure. LUNAPARK1 deletion in rtn1Δrtn2Δyop1Δ cells restores the pnER/cER functional asymmetry, tubular ER generation, and ER inheritance blocks. Thus, Reticulon/Yop1-dependent changes in ER structure are linked to ER inheritance during the yeast cell cycle. Segregation of functional organelles during the cell cycle is crucial to generate healthy daughter cells. In Saccharomyces cerevisiae, ER stress causes an ER inheritance block to ensure cells inherit a functional ER. Here, we report that formation of tubular ER in the mother cell, the first step in ER inheritance, depends on functional symmetry between the cortical ER (cER) and perinuclear ER (pnER). ER stress induces functional asymmetry, blocking tubular ER formation and ER inheritance. Using fluorescence recovery after photobleaching, we show that the ER chaperone Kar2/BiP fused to GFP and an ER membrane reporter, Hmg1-GFP, behave differently in the cER and pnER. The functional asymmetry and tubular ER formation depend on Reticulons/Yop1, which maintain ER structure. LUNAPARK1 deletion in rtn1Δrtn2Δyop1Δ cells restores the pnER/cER functional asymmetry, tubular ER generation, and ER inheritance blocks. Thus, Reticulon/Yop1-dependent changes in ER structure are linked to ER inheritance during the yeast cell cycle. Eukaryotic cells possess many regulatory and cell-cycle checkpoints to ensure proper DNA replication and segregation during the cell cycle (Rhind and Russell, 2012Rhind N. Russell P. Signaling pathways that regulate cell division.Cold Spring Harb. Perspect. Biol. 2012; 4: a005942Crossref Scopus (109) Google Scholar, Lara-Gonzalez et al., 2012Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, Yasutis and Kozminski, 2013Yasutis K.M. Kozminski K.G. Cell cycle checkpoint regulators reach a zillion.Cell Cycle. 2013; 12: 1501-1509Crossref PubMed Scopus (47) Google Scholar). The loss of such control is an underlying cause of many human diseases, including cancer (Abbas et al., 2013Abbas T. Keaton M.A. Dutta A. Genomic instability in Cancer.Cold Spring Harb. Perspect. Biol. 2013; 5: a012914Crossref Scopus (123) Google Scholar). In contrast, less is known about the regulatory pathways governing inheritance of cytoplasmic components, and few studies have investigated how cell-cycle checkpoints ensure transmission of functional organelles, such as the ER, to daughter cells. After translation, linear polypeptides of secretory proteins are translocated into the ER lumen for chaperone-assisted folding and post-translational modifications before exiting the ER (Ron and Walter, 2007Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4863) Google Scholar, Rutkowski and Kaufman, 2004Rutkowski D.T. Kaufman R.J. A trip to the ER: coping with stress.Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). When folding demand exceeds ER capacity, known as ER stress, three ER transmembrane protein sensors (IRE1, PERK, and ATF6) initiate the unfolded protein response (UPR) (Walter and Ron, 2011Walter P. Ron D. The unfolded protein response: from stress pathway to homeostatic regulation.Science. 2011; 334: 1081-1086Crossref PubMed Scopus (3907) Google Scholar). The UPR re-establishes ER homeostasis by upregulating the transcription of genes encoding ER chaperones, protein folding and modifying components, and lipid-generating enzymes (Mcmaster, 2001Mcmaster C.R. Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery.Biochem. Cell Biol. 2001; 79: 681-692Crossref PubMed Scopus (70) Google Scholar). Importantly, the ER cannot be synthesized de novo and arises only from pre-existing ER, implying that regulatory mechanisms must exist to regulate its inheritance during the cell cycle. We previously identified a cell-cycle surveillance mechanism in Saccharomyces cerevisiae, termed the ERSU (ER Stress Surveillance) pathway that operates during ER stress to ensure daughter cells inherit functional ER (Babour et al., 2010Babour A. Bicknell A.A. Tourtellotte J. Niwa M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance.Cell. 2010; 142: 256-269Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The ERSU pathway operates independently of the UPR; instead, the ERSU is centrally regulated by the Slt2 mitogen-activated protein (MAP) kinase. During ER stress, slt2Δ cells fail to relocalize the septin ring away from the bud neck and the stressed ER enters the daughter cell, ultimately causing death. However, slt2Δ cell growth is rescued by preventing stressed ER entry into the daughter cell, showing that inheritance of stressed ER is the major cause of slt2Δ cell death during ER stress. The yeast ER exists as two major subdomains: the perinuclear ER (pnER), which surrounds the nucleus, and the cortical ER (cER), which is located at the periphery of the cell in close contact with the plasma membrane. Although the two subdomains are contiguous and physically connected by tubules, they adopt different structures. While the pnER is sheet-like and continuous with the nuclear envelope, the cER is a more distinct structure consisting of interconnected tubules (Hu et al., 2011Hu J. Prinz W.A. Rapoport T.A. Weaving the web of ER tubules.Cell. 2011; 147: 1226-1231Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Friedman and Voeltz, 2011Friedman J.R. Voeltz G.K. The ER in 3D: a multifunctional dynamic membrane network.Trends Cell Biol. 2011; 21: 709-717Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, De Martin et al., 2005De Martin P.E. Novick P. Ferro-Novick S. The organization, structure, and inheritance of the ER in higher and lower eukaryotes.Biochem. Cell Biol. 2005; 83: 752-761Crossref PubMed Scopus (24) Google Scholar). The mammalian ER also contains sheet-like structures (cisternae) and reticular ER. The ER sheets are connected by a network of polygonal tubules generated from three-way junctions of tubular membranes that extend close to the plasma membrane (English et al., 2009English A.R. Zurek N. Voeltz G.K. Peripheral ER structure and function.Curr. Opin. Cell Biol. 2009; 21: 596-602Crossref PubMed Scopus (137) Google Scholar, Goyal and Blackstone, 2013Goyal U. Blackstone C. Untangling the web: mechanisms underlying ER network formation.Biochim. Biophys. Acta. 2013; 1833: 2492-2498Crossref PubMed Scopus (112) Google Scholar). They are covered by abundant ribosomes and play a key role in the production of secretory proteins. In yeast and mammalian cells, the formation and maintenance of tubular ER requires several proteins: the reticulons and DP1/Yop1, which stabilize the highly curved tubular ER structure (Voeltz et al., 2006Voeltz G.K. Prinz W.A. Shibata Y. Rist J.M. Rapoport T.A. A class of membrane proteins shaping the tubular endoplasmic reticulum.Cell. 2006; 124: 573-586Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar); members of the dynamin-related GTPase family such as Atlastin/Sey1 (Wang et al., 2013Wang S. Romano F.B. Field C.M. Mitchison T.J. Rapoport T.A. Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts.J. Cell Biol. 2013; 203: 801-814Crossref PubMed Scopus (68) Google Scholar, Anwar et al., 2012Anwar K. Klemm R.W. Condon A. Severin K.N. Zhang M. Ghirlando R. Hu J. Rapoport T.A. Prinz W.A. The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae.J. Cell Biol. 2012; 197: 209-217Crossref PubMed Scopus (87) Google Scholar): and antagonistic proteins such as Lunapark1 (Chen et al., 2012Chen S. Novick P. Ferro-Novick S. ER network formation requires a balance of the dynamin-like GTPase Sey1p and the Lunapark family member Lnp1p.Nat. Cell Biol. 2012; 14: 707-716Crossref PubMed Scopus (103) Google Scholar). How the cell controls the dynamic ratio of sheet-like and tubular ER structures is currently unknown. Despite their complexity, both ER subdomains are present in newly generated cells. In yeast, an initial ER tubule emerges from the mother cell pnER, moves along the mother-daughter axis, enters the daughter cell, and then anchors at the bud tip before spreading around the periphery of the daughter cell (Fehrenbacher et al., 2002Fehrenbacher K.L. Davis D. Wu M. Boldogh I. Pon L.A. Endoplasmic reticulum dynamics, inheritance, and cytoskeletal interactions in budding yeast.Mol. Biol. Cell. 2002; 13: 854-865Crossref PubMed Scopus (69) Google Scholar). In an elegant study using electron tomography, West et al., 2011West M. Zurek N. Hoenger A. Voeltz G.K. A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature.J. Cell Biol. 2011; 193: 333-346Crossref PubMed Scopus (252) Google Scholar also showed that tubular ER can emerge from the mother cell pnER, suggesting that this is the initial event for ER inheritance in S. cerevisiae. The distinct origins and activities of the pnER and cER described above raise the possibility that the differential functional status of the two ER subdomains might be critical to ER tubule formation and ER inheritance under both normal and ER stress conditions. Here, we addressed this question by examining differences in pnER and cER function and its relationship to ER tubule formation and the block in ER inheritance during ER stress. We previously showed that, in yeast ER, stress blocks cER inheritance, but the pnER is transmitted normally to the daughter cell (Figures 1A and S1A) (Babour et al., 2010Babour A. Bicknell A.A. Tourtellotte J. Niwa M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance.Cell. 2010; 142: 256-269Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). These findings suggested that ER stress inducers might have different effects on the cER and pnER. To investigate this, we analyzed the dynamics of Kar2/BiP-sfGFP, a major ER luminal chaperone, using fluorescence recovery after photobleaching (FRAP) assays (Lajoie et al., 2012Lajoie P. Moir R.D. Willis I.M. Snapp E.L. Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells.Mol. Biol. Cell. 2012; 23: 955-964Crossref PubMed Scopus (58) Google Scholar, Lai et al., 2010Lai C.W. Aronson D.E. Snapp E.L. BiP availability distinguishes states of homeostasis and stress in the endoplasmic reticulum of living cells.Mol. Biol. Cell. 2010; 21: 1909-1921Crossref PubMed Scopus (58) Google Scholar). In response to ER stress, Kar2/BiP binding to unfolded client proteins increases, reducing its mobility within the ER lumen (Snapp et al., 2006Snapp E.L. Sharma A. Lippincott-Schwartz J. Hegde R.S. Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 6536-6541Crossref PubMed Scopus (89) Google Scholar). Therefore, the rate of Kar2-sfGFP FRAP in the pnER or cER is a direct measure of Kar2/BiP mobility, and thus of the ER stress level in that compartment. To induce ER stress, we treated wild-type (WT) yeast cells with tunicamycin (Tm), an N-glycosylation inhibitor that causes accumulation of unglycosylated unfolded proteins in the ER, and photobleached discrete regions of the cER or pnER, and monitored fluorescence recovery over time. Tm-treated cells showed delayed fluorescence recovery in both the cER and pnER compared with DMSO-treated cells, although the magnitude and kinetics of the effect were markedly different in the two compartments (Figures 1B–1E). The reduced rate of Kar2-sfGFP fluorescence recovery was detected in the cER within 30 min of Tm treatment, and the effect was further increased in cells exposed to Tm for 3 hr (Figures 1B and 1D). In contrast, Tm had little effect on Kar2-sfGFP mobility in the pnER at 30 min, and a small but significant reduction in mobility was noted only after 3 hr incubation with Tm (Figures 1C and 1E). Kar2-sfGFP mobility in the cER and pnER of DMSO-treated cells was essentially identical (Figures 1B–1E). These experiments suggested a fundamental difference in the effects of ER stress on the behavior of the same chaperone protein in the cER and pnER. Previously, we reported that the ERSU and UPR pathways are distinct and that the UPR was not involved in the ER stress-induced ER inheritance block (Babour et al., 2010Babour A. Bicknell A.A. Tourtellotte J. Niwa M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance.Cell. 2010; 142: 256-269Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Activation of the UPR requires the ER transmembrane receptor kinase/endoribonuclease Ire1, whereas the MAP kinase Slt2 activates the ERSU pathway, which does not require Ire1 (Babour et al., 2010Babour A. Bicknell A.A. Tourtellotte J. Niwa M. A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance.Cell. 2010; 142: 256-269Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To test whether Kar2-sfGFP mobility in the cER and pnER is differentially regulated by the ERSU and UPR pathways, we performed FRAP assays in ire1Δ and slt2Δ cells (Figures 1F–1I, S1B, and S1C). There were no differences in the cER or pnER Kar2-sfGFP fluorescence recovery of unstressed WT, slt2Δ, and ire1Δ cells (Figures 1D–1I, DMSO). Kar2-sfGFP mobility was also similar in the cER of Tm-treated WT, slt2Δ, and ire1Δ cells (Figures 1D, 1F, and 1H). However, a striking difference was noted in the pnER analyses: Tm treatment for 3 hr caused a marked reduction in Kar2-sfGFP mobility in the pnER of slt2Δ cells but not WT or ire1Δ cells (Figures 1E, 1G, and 1I). In slt2Δ cells, Tm had very similar effects on Kar2/BiP-sfGFP mobility in the cER and pnER (Figures 1H and 1I). Since slt2Δ cells do not block ER inheritance under ER stress conditions, unlike WT and ire1Δ cells, these data point to a potential link between the functional state of the pnER and the ability to halt ER inheritance during ER stress. To ensure that Kar2-sfGFP fluorescence recovery faithfully reflects its association with unfolded proteins, we performed similar FRAP experiments with cells expressing kar2-1, a temperature-sensitive KAR2 mutation that disrupts its ability to bind unfolded proteins (Kabani et al., 2003Kabani M. Kelley S.S. Morrow M.W. Montgomery D.L. Sivendran R. Rose M.D. Gierasch L.M. Brodsky J.L. Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP.Mol. Biol. Cell. 2003; 14: 3437-3448Crossref PubMed Scopus (87) Google Scholar) (Figures S2A and S2B). Indeed, there was little difference in kar2-1-sfGFP fluorescence recovery in the pnER of control and ER-stressed cells, but there was a small but notable delay in recovery detected in the cER of stressed cells (Figures S2A and S2B). This observation suggested that at least a portion of the delay in Kar2-sfGFP fluorescence recovery was due to an unknown, chaperone-independent effect of ER stress on mobility. To further test this idea, we examined the mobility of Hmg1-GFP, a fusion protein carrying a single ER transmembrane domain of the non-chaperone ER protein Hmg1 (Hampton et al., 1996Hampton R.Y. Koning A. Wright R. Rine J. In vivo examination of membrane protein localization and degradation with green fluorescent protein.Proc. Natl. Acad. Sci. USA. 1996; 93: 828-833Crossref PubMed Scopus (106) Google Scholar). As we observed with the mutant kar2-1-sfGFP protein, ER stress (Tm) caused a small delay in Hmg1-GFP fluorescence recovery in the cER, but not in the pnER (Figures 1J, 1K, and S2C). While the relationship between the FRAP response and functional status of ER chaperones has been well documented, the kar2-1-sfGFP and Hmg1-GFP results suggest that a small portion of the delay in Kar2-sfGFP fluorescence recovery in the stressed cER is independent of its chaperone activity and instead reflects the ER status. Collectively, these experiments demonstrate that ER stress has differential effects on the cER and pnER, and that the loss of Slt2 affects only the function of the pnER. The differential mobility of the same protein in the cER and pnER during ER stress was surprising because the two compartments are thought to be contiguous and interconnected; therefore, Kar2/BiP is expected to travel freely throughout the network. To examine if ER stress causes a disconnection between the pnER and cER, we performed fluorescence loss in photobleaching (FLIP) experiments. We found that after repeatedly photobleaching a small region of the cER, Kar2-sfGFP fluorescence in the pnER and a remote region of the cER decayed at a similar rate in DMSO- and Tm-treated cells, and eventually all fluorescence was lost (Figures 2A and 2B ). These data demonstrate that the pnER and cER remain interconnected during ER stress and that Kar2-sfGFP mobility differences in the two compartments are not due to physical separation. Two potential scenarios could explain why ER stress did not affect Kar2/BiP fluorescence recovery in the pnER: (1) the pnER could contain significantly fewer unfolded proteins than the cER, and (2) unfolded proteins could be generated in the pnER, but Kar2-sfGFP might not dissociate from Ire1 and thus be unable to bind unfolded proteins, as suggested by a recent study (Ishiwata-Kimata et al., 2013Ishiwata-Kimata Y. Promlek T. Kohno K. Kimata Y. BiP-bound and nonclustered mode of Ire1 evokes a weak but sustained unfolded protein response.Genes Cells. 2013; 18: 288-301Crossref PubMed Scopus (23) Google Scholar). If true, the latter scenario would suggest that pnER-localized and cER-localized Kar2-sfGFP acquire different properties during ER stress. To test the first possibility, we examined the formation of CPY∗-mRFP or GFP-CFTR protein aggregates in each ER subdomain during ER stress (Kakoi et al., 2013Kakoi S. Yorimitsu T. Sato K. COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.Mol. Biol. Cell. 2013; 24: 633-642Crossref PubMed Scopus (14) Google Scholar, Fu and Sztul, 2003Fu L. Sztul E. Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.J. Cell Biol. 2003; 160: 157-163Crossref PubMed Scopus (57) Google Scholar, Pina and Niwa, 2015Pina F.J. Niwa M. The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance.Elife. 2015; 4https://doi.org/10.7554/eLife.06970Crossref PubMed Scopus (17) Google Scholar). CPY∗-mRFP aggregates activate both the UPR and ERSU pathways, whereas GFP-CFTR aggregates do not (Pina and Niwa, 2015Pina F.J. Niwa M. The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance.Elife. 2015; 4https://doi.org/10.7554/eLife.06970Crossref PubMed Scopus (17) Google Scholar). This difference allows us to observe the cER and pnER localization pattern of aggregates that do or do not induce ER stress. In both CPY∗-mRFP- and CFP-CFTR-expressing cells, approximately twice as many foci were present in the pnER than in the cER. This ratio did not change in the presence of Tm, indicating that unfolded proteins are abundant in the pnER and that the ratio of protein aggregates in the pnER to the cER does not decrease under ER stress (Figures 2C and 2D). To test the second possibility, we monitored the distribution of Ire1 in the cER and pnER to examine UPR activation. During ER stress, activated Ire1 is released from Kar2/BiP and autophosphorylates, forming oligomers that can be detected as foci in cells expressing Ire1-GFP (Aragon et al., 2009Aragon T. Van Anken E. Pincus D. Serafimova I.M. Korennykh A.V. Rubio C.A. Walter P. Messenger RNA targeting to endoplasmic reticulum stress signalling sites.Nature. 2009; 457: 736-740Crossref PubMed Scopus (253) Google Scholar, Kimata et al., 2007Kimata Y. Ishiwata-Kimata Y. Ito T. Hirata A. Suzuki T. Oikawa D. Takeuchi M. Kohno K. Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins.J. Cell Biol. 2007; 179: 75-86Crossref PubMed Scopus (225) Google Scholar). Thus, the presence of Ire1 foci is a measure of Kar2/BiP-Ire1 dissociation. Using DsRed-HDEL as an ER reporter, we found that Ire1-GFP was distributed throughout the ER in unstressed cells, but discrete Ire1-GFP foci were evident in both the pnER and cER within 1 hr and persisted for at least 3 hr after Tm treatment (Figure 2E). Furthermore, Ire1-GFP foci were twice as abundant in the pnER as in the cER, as was observed for CYP∗ and CFTR aggregates. Taken together, these observations indicate that ER stress-induced protein aggregates are abundant in the pnER and that the difference in Kar2/BiP mobility in the two compartments during ER stress does not reflect a lack of unfolded proteins or a perceived lack of ER stress in the pnER. Our results so far indicate that the differential effect of ER stress on the cER and pnER functional state, as reflected by Kar2-sfGFP mobility, was dependent on the ERSU (Slt2), independent of the UPR (Ire1) and was not caused by differences in unfolded proteins levels or a physical disconnection between the cER and pnER. We next hypothesized that ER stress might induce distinct structural changes in the two ER domains. Indeed, there are known differences in structural elements in the pnER and cER. For example, certain areas of the cER directly connect with the plasma membrane (PM) via tethering proteins such as lst2, Tcb1, Tcb2, Tcb3, Scs2, Scs22 (Manford et al., 2012Manford A.G. Stefan C.J. Yuan H.L. Macgurn J.A. Emr S.D. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.Dev. Cell. 2012; 23: 1129-1140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), whereas the pnER does not form such contacts. Therefore, we examined Kar2-sfGFP mobility in a yeast strain lacking lst2, Tcb1, Tcb2, Tcb3, Scs2, Scs22, labeled Δtether. In the Δtether strain, the cER structure is severely altered such that it is no longer juxtaposed to the PM but is present in the middle of the cytoplasm (Figure S3A), as reported (Manford et al., 2012Manford A.G. Stefan C.J. Yuan H.L. Macgurn J.A. Emr S.D. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.Dev. Cell. 2012; 23: 1129-1140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). However, we found that, like WT cells, Kar2-sfGFP mobility in the pnER of Δtether cells remained high compared with that in the cER (Figures 3A–3D and S3B). Similar results were found using Δtether cells expressing Hmg1-GFP (Figures 3E–3H). Finally, stressed Δtether and WT cells blocked ER inheritance similarly (Figures 3I–3J). These findings indicate that the different cER and pnER functional responses cannot be explained by tethering protein-dependent structural differences, and that the tethering proteins are not required for the ERSU pathway. As described earlier, the ER network is composed of tubules and sheets. In both yeast and mammalian cells, ER sheets are found juxtaposed to the nucleus, whereas tubular ER is peripherally located away from the nucleus and close to the PM. The high membrane curvature of the ER is stabilized by two reticulon proteins, Rtn1 and Rtn2, and a reticulon-like protein, DP1/Yop1 (Stefano et al., 2014Stefano G. Hawes C. Brandizzi F. ER - the key to the highway.Curr. Opin. Plant Biol. 2014; 22: 30-38Crossref PubMed Scopus (48) Google Scholar, Chiurchiu et al., 2014Chiurchiu V. Maccarrone M. Orlacchio A. 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To determine whether an intact cER tubular structure is necessary to establish ER stress-induced functional asymmetry between the cER and pnER, we examined Kar2-sfGFP mobility in cells lacking RTN1, RTN2, and YOP1. rtn1Δrtn2Δyop1Δ cells form extended sheets of cER, rather than the fine reticular structure seen in WT cells, which remain juxtaposed to the PM (Figure S3A) (Voeltz et al., 2006Voeltz G.K. Prinz W.A. Shibata Y. Rist J.M. Rapoport T.A. A class of membrane proteins shaping the tubular endoplasmic reticulum.Cell. 2006; 124: 573-586Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, De Craene et al., 2006De Craene J.O. Coleman J. Estrada De Martin P. Pypaert M. Anderson S. Yates 3rd, J.R. Ferro-Novick S. Novick P. Rtn1p is involved in structuring the cortical endoplasmic reticulum.Mol. Biol. Cell. 2006; 17: 3009-3020Crossref PubMed Scopus (105) Google Scholar, Hu et al., 2008Hu J. Shibata Y. Voss C. Shemesh T. Li Z. Coughlin M. Kozlov M.M. Rapoport T.A. 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In contrast to WT cells, we found that Kar2-sfGFP fluorescence recovery decreased to similar extents in the pnER and cER of Tm-treated rtn1Δrtn2Δyop1Δ cells, closely resembles the phenotype of slt2Δ cells (Figures 4A–4C , 4F–H, and S3C). Kar2-sfGFP mobility decreased similarly in the cER of stressed WT and rtn1Δrtn2Δyop1Δ cells (Figures 4F, 4H, and S3C). Similar results were obtained with the Hmg1-GFP reporter in rtn1Δrtn2Δyop1Δ and slt2Δ cells (Figures S4A and S4B), indicating that the mobility of chaperone and non-chaperone pnER-resident proteins was affected. These data point to a central role for the reticulons and DP1/Yop1 in dictating how the function of the cER and pnER is affected by ER stress. Furthermore, the data indicate that ER stress has effects on ER function and inheritance that go beyond influencing the behavior of chaperones in the lumen and extend to altering the structure and/or composition of the ER. Since we observed a similar reduction in Kar2/BiP mobility in the pnER of stressed slt2Δ cells and rtn1Δrtn2Δyop1Δ cells (Figures 4B, 4C, 4G, and 4H), we asked whether rtn1Δrtn2Δyop1Δ cells had also lost the ability to prevent transmission of a dysfunctional ER to the daughter cells. Compared with WT cells, ER inheritance in rtn1Δrtn2Δyop1Δ cells was reduced even under normal growth conditions, indicating the importance of reticulons for normal ER inheritance. However, ER stress had no further effect on ER inheritance in these cells (Figures 4K and 4L), a phenotype also observed in stressed slt2Δ cells (Figure 4M). These observations revealed an intriguing correlation between Kar2/BiP mobility in the pnER and the ability to block cER inheritance in response to ER stress. To probe this further, we examined the association between the function of the pnER and its ability to form ER tubules. For this, we quantified the appearance of tubular ER from the pnER in WT, slt2Δ, and rtn1Δrtn2Δyop1Δ cells. While more than 80% of unstressed WT cells showed evidence of early tubular ER formation, ER stress reduced this to ∼30%, consistent with the block in ER inheritance under these conditions (Figures 4K," @default.
• W2336735095 created "2016-06-24" @default.
• W2336735095 creator A5030069081 @default.
• W2336735095 creator A5044562249 @default.
• W2336735095 creator A5045874120 @default.
• W2336735095 creator A5062514487 @default.
• W2336735095 date "2016-05-01" @default.
• W2336735095 modified "2023-10-15" @default.
• W2336735095 title "Reticulons Regulate the ER Inheritance Block during ER Stress" @default.
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