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Results: 8

1.
FIGURE 4:

FIGURE 4:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The relationship between the components of the endoplasmic reticulum and ERAC structures. Log-phase cultures of wild-type cells (ANY21) coexpressing EGFP-CFTR (induced as described in Figure 1A) and Sar1p-mCherry (A), Sar1p-D32G-mCherry (B), Sec71p-mCherry (C), Sec23p-mCherry (D), or Sec13p-mCherry (E) were visualized by confocal microscopy at 23ºC, except B, which was observed at 37ºC.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
2.
FIGURE 5:

FIGURE 5:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The diacidic ER export motif of CFTR is dispensable for its entry into ERACs. Wild-type (ANY21) cells containing the EGFP-CFTR plasmid (top) or EGFP-CFTR-D567A plasmid (bottom) were grown to mid–log phase at 23ºC and incubated with copper-containing medium for 2 h to induce expression. The cells were then visualized by confocal microscopy.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
3.
FIGURE 3:

FIGURE 3:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The effect of overexpressing individual COPII components on ERAC formation in temperature-sensitive mutants defective in COPII vesicle formation. (A–C) The transformants used in Figure 2 were grown to mid–log phase and treated with copper at 23ºC (left) or 37ºC (right) for 2 h to induce EGFP-CFTR expression. ERAC formation was quantified as described in Figure 1B. The experiments were repeated at least three times, and >70 cells were analyzed for each condition.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
4.
FIGURE 7:

FIGURE 7:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

EGFP-CFTR forms a complex with Hlj1p in the ER. Microsomes (100 μg) expressing EGFP-CFTR (induced for 2 h) with (+) or without (–) GST-Hlj1p were solubilized with 1% Triton X-100. The resulting extract was cleared by centrifugation, and the supernatant was incubated with glutathione–Sepharose beads. The beads were washed, and proteins were released in the presence of 10 mM glutathione. The released proteins were analyzed by SDS–PAGE, followed by immunoblotting with antibodies to GFP, Sec23p, Sar1p, Sec12p, Sec61p, and GST. The total lane (T) represents 10% of the starting microsomal extract used in the lane labeled (+).

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
5.
FIGURE 8:

FIGURE 8:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

Hsp40s and COPII components act in the same pathway to drive ERAC formation. (A) hlj1∆ cells coexpressing EGFP-CFTR (induced as described in Figure 1A) and mCherry-Hlj1p were observed by confocal microscopy. (B) hlj1∆ cells harboring pEGFP-CFTR were transformed with pYO324 (2 μ vector), pSAR1 (2 μ SAR1), pSEC12 (2 μ SEC12), pSEC23 (2 μ SEC23), pSEC16 (2 μ SEC16), pSEC13 (2 μ SEC13), or pSAR1D32G (2 μ SAR1D32G), and the transformants were grown to mid–log phase and induced with copper at 23ºC for 2 h to induce EGFP-CFTR expression. ERAC formation was quantified as described in Figure 1B. The experiments were repeated at least three times, and >150 cells were analyzed for each condition.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
6.
FIGURE 1:

FIGURE 1:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The formation of EGFP-CFTR–induced ERACs requires COPII function. (A) Wild-type (ANY21) cells containing the EGFP-CFTR expression plasmid were grown to mid–log phase at 23ºC and incubated with copper-containing medium for 2 h to induce expression, followed by observation with confocal microscopy. (B) Wild-type and sec12-4, sec23-1, sec13-1, sec16-2, sar1D32G, sec18-1, and sec17-1 strains were grown to mid–log phase and induced with copper at 23ºC (top) or 37ºC (bottom) for 2 h to induce EGFP-CFTR expression. ERAC formation is quantified using the three categories that were defined in A, and the data are presented as the percentage of cells expressing EGFP-CFTR. The experiments were repeated at least three times, and >100 cells were analyzed for each condition.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
7.
FIGURE 6:

FIGURE 6:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The formation of EGFP-CFTR–induced ERACs is ER associated and Hsp40 dependent. (A) ydj1∆ and hlj1∆ cells containing the EGFP-CFTR plasmid were grown to mid–log phase at 23ºC and incubated with copper-containing medium for 2 h to induce EGFP-CFTR expression, followed by observation with confocal microscopy. (B) Wild-type, hlj1∆, ydj1∆, and apj1∆ cells harboring pEGFP-CFTR were grown to mid–log phase and induced with copper at 23ºC for 2 h to induce EGFP-CFTR expression. ERAC formation was quantified as described in Figure 1B. The experiments were repeated at least three times, and >50 cells were analyzed for each condition. (C) Wild-type, hlj1∆, and ydj1∆ cells expressing EGFP-CFTR were subjected to cycloheximide chase analysis as described in Materials and Methods, and the amount of EGFP-CFTR at time point 0 was set to 1.0. The relative amounts of EGFP-CFTR remaining in wild-type (open circles), hlj1∆ (closed squares), and ydj1∆ (closed triangles) strains over time were plotted.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.
8.
FIGURE 2:

FIGURE 2:. From: COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments.

The effect of overexpressing individual COPII components on the growth of temperature-sensitive mutants defective in COPII vesicle formation. (A) sec23-1 (MBY8-20C), sec12-4 (MBY10-7A), and sec16-2 (MBY4-1A) cells harboring pEGFP-CFTR were transformed with pYO324 (2 μ vector), pSAR1 (2 μ SAR1), pSEC12 (2 μ SEC12), pSEC23 (2 μ SEC23), pSEC16 (2 μ SEC16), or pSEC13 (2 μ SEC13), and the transformants were grown to saturation at 23ºC in MCD medium lacking uracil and tryptophan (MCD-Ura-Trp). The cells were diluted to a final concentration of 0.3 at OD600, and 5 μl of 10-fold dilutions was spotted onto MCD-Ura-Trp plates. The plates were incubated at 23 or 37ºC as indicated. (B) sec23-1 (MBY8-20C) cells transformed with pYO324 (2 μ vector) or pSEC16(2 μ SEC16) were grown at 23 or 37ºC in selective media. Equal amounts of whole-cell extracts were subjected to immunoblotting with anti-CPY antibody. The erv29∆ strain was used as a control. The ER (p1) and mature (m) forms of CPY are indicated.

Shogo Kakoi, et al. Mol Biol Cell. 2013 March 1;24(5):633-642.

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