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. 2018 Jul;9(7):616-628.
doi: 10.1007/s13238-017-0492-5. Epub 2017 Nov 22.

Sec61β Facilitates the Maintenance of Endoplasmic Reticulum Homeostasis by Associating Microtubules

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Free PMC article

Sec61β Facilitates the Maintenance of Endoplasmic Reticulum Homeostasis by Associating Microtubules

Yimeng Zhu et al. Protein Cell. .
Free PMC article

Abstract

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.

Keywords: ER stress; Microtubule; Ribosome; Sec61β; Translocon.

Figures

Figure 1
Figure 1
Overexpressed Sec61β induces bundling of the ER and microtubule. (A) COS-7 cells transfected with Sec61β-HA were immunostained for HA (green) and endogenous tubulin or luminal ER protein, calreticulin (red), and visualized by fluorescent confocal microscopy. Insets show the enlargement of the indicated area. (B) As in (A), but with RFP-Sec61β-HA and were immunostained only for tubulin or calreticulin (green). (C) As in (A), but wild type COS-7 cells were immunostained for calreticulin (green) and tubulin (red). (D) COS-7 cells co-transfected with Sec61β, ER-DsRed (red), and mEmerald-Ensconsin (green) were visualized live by fluorescent confocal microscopy. (E) Domain structures of constructs. (F) As in (A), but with various constructs shown in (E), and were immunostained for HA-epitope (green) and tubulin (red). (G) The ER morphology of samples shown in (A) and (B) was categorized as “bundled” and “normal” respectively. A total of 150 cells were counted for each sample. All graphs were representative of three repetitions. In (A–D) and (F), scale bars are 10 μm
Figure 2
Figure 2
Sec61β interacts with microtubule. (A) COS-7 cells were harvested for microtubule co-sedimentation in the absence or presence of GTP/paclitaxel (G/P). Samples of the supernatant (S) and pellet (P) were analyzed by Western blotting. (B–D) As in (A), but transfected with full length Sec61β (B), Sec61β lacking residues 20–44 (C) or RFP-tagged Sec61β (D). The bands with asterisk (*) may be degraded RFP-Sec61β-HA. (E) Purified wild-type HA-cytSec61β was incubated with microtubules (MT) or not, for microtubule co-sedimentation. Samples were analyzed by Western blotting. (F) As in (E), but with HA-cytSec61β lacking residues 20–44. (G) Purified wild-type HA cytSec61β or HA-cytSec61β lacking residues 20–44 was incubated with tubulins and precipitated with anti-HA antibody. The levels of indicated proteins were analyzed with Western blotting
Figure 3
Figure 3
Depletion of Sec61β triggers ER stress. (A) COS-7 cells were infected with shRNA-expressing viruses. The levels of Sec61β were determined by Western blotting, and the unspliced (U) XBP1 and spliced (S) XBP1 by RT-PCR of XBP1 mRNA were resolved by agarose gel. ER stress markers, including Bip and phosphorylated eIF2α, were analyzed by Western blotting. Asterisk (*) indicates a nonspecific band. (B) The unspliced (U) XBP1 and spliced (S) XBP1 by RT-PCR of XBP1 mRNA from infected cells or thapsigargin (TG) treated cells were resolved by agarose gel. (C) U2OS cells were transfected with siRNAs of Sec61β for 48 h or 96 h. The levels of Sec61β were determined by Western blotting, and the unspliced XBP1 and spliced XBP1 by RT-PCR of XBP1 mRNA were resolved by agarose gel
Figure 4
Figure 4
Sec61β-regulated ER homeostasis requires microtubule binding. (A) SiRNA transfected Flp-In-293 cells were treated with tetracycline as indicated. The samples were analyzed by Western blotting and agarose gel. (B) As in (A), but in cells expressing MTBClimp63-TMSec61β-HA and RFP-Sec61β-HA respectively. The bands with asterisk (*) may be degraded RFP-Sec61β-HA
Figure 5
Figure 5
The function of Sec61β is essential and conserved in C. elegans. (A) Fluorescent micrographs of hypodermal cells in gfp::tram-1 worms expressing CHERRY::Y38F2AR.9. DIC, differential interference contrast. Scale bar: 5 μm. (B) The y38f2ar.9 RNAi was injected into worms carrying Phsp-4::GFP, and GFP fluorescence was assessed. For rescue assay, y38f2ar.9 RNAi was injected into Phsp-4::GFP ER stress reporter worms carrying human SEC61B, MTBClimp63-TMSec61β or RFP-hSEC61B, and GFP fluorescence was assessed. Scale bar: 100 μm
Figure 6
Figure 6
Depletion of Sec61β improves the lateral mobility of translocon complexex. (A) M3/18 cells transfected with siRNA of Sec61β or Climp63 for 48 h were lyzed for analysis after FRAP. The levels of Sec61β were determined by Western blotting, with actin as a loading control. Climp63 and Actin by RT-PCR were resolved by agarose gel. (B) M3/18 cells in (A) were analyzed by fluorescence recovery after photobleaching assay. The initial fluorescence intensity was set at 100%. Data are mean ± SEM. (n =7 for control group, 6 for siSEC61B group and 5 for siClimp63 group). (C) M3/18 cells were transfected with siRNA of Sec61β for 48 h. The lysates were precipitated with anti-GFP antibody. The levels of indicated proteins were analyzed with Western blotting
Figure 7
Figure 7
Sec61β stabilizes membrane attachment of ribosomes. (A) Polysome profiles of U2OS cells transfected control siRNA (siCtrl) or siSEC61B#2. (B) Total lysates from (A) were analyzed by Western blotting, with actin as a loading control. (C) As in (A), but U2OS cells co-transfected siSEC61B#2 and indicated plasmids. (D) As in (B), but from cells in (C). For all of them, the trends are similar among at least two independent repeats

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