Figure 3. Localization of the... | Rockefeller University Press

Figure 3.

$Figure 3. Localization of the fluorescent secretory cargo after ER export. (A) Colocalization of the cargo with Golgi and PVE markers soon after ER export. Cells expressing the APVNTT-DsRed-Express2-FKBPRD(C22V) cargo and either GFP-Vrg4 (early Golgi), Sec7-GFP (late Golgi), or Vps8-GFP (PVE) in a pdr1Δ pdr3Δ or pdr1Δ pdr3Δ vps10-104 background were grown to mid-log phase, treated with SLF for 5 min, fixed, and imaged by confocal microscopy. Shown are representative projected Z-stacks. Arrowheads indicate examples of structures containing detectable cargo, and arrows indicate examples of structures lacking detectable cargo. Scale bar, 2 µm. (B) Quantification from A of the percentages of labeled structures containing detectable cargo. Data were obtained from at least 40 cells per condition. Error bars indicate SEM. (C) Immunoblot of cell-associated and secreted cargo after SLF addition in rich medium. Yeast were grown to mid-log phase in YPD, washed twice with fresh YPD, and treated with SLF. At the indicated time points, cell-associated and secreted fractions were separated by centrifugation, and then the sedimented cells were lysed and proteins from both fractions were precipitated. Samples were subjected to endo H treatment followed by SDS-PAGE and immunoblotting for FKBP. The visualized band corresponds to the upper band in the “+” lane of D. (D) Immunoblot demonstrating hyperglycosylation of the secreted cargo. Proteins were precipitated from rich medium 60 min after SLF addition. Samples were then treated or mock treated with endo H, followed by SDS-PAGE and immunoblotting for FKBP. A high-molecular-weight smear was seen only in the absence of endo H treatment. In the endo H–treated sample, the upper band is the full-length fusion protein, and the lower band is a minor species that was exaggerated by overloading the gel. The lower band might have been generated by Kex2 protease cleavage (Rockwell et al., 2002) of FKBP after the KR dipeptide at positions 17–18. MW, molecular weight marker, with the sizes of the reference proteins shown at the left. (E) Visualization of secreted cargo trapped in the periplasm in minimal medium. Cells expressing the aggregated cargo and the ER marker Erg11-GFP were grown to mid-log phase in NSD buffered at pH 6, then treated with SLF for 30 min and mounted in a flow chamber. Unbuffered NSD (pH ∼4) was flowed over the cells while imaging with a confocal microscope. Shown are projected Z-stacks from Video 2. Scale bar, 2 µm.$

Localization of the fluorescent secretory cargo after ER export. (A) Colocalization of the cargo with Golgi and PVE markers soon after ER export. Cells expressing the APVNTT-DsRed-Express2-FKBP^RD(C22V) cargo and either GFP-Vrg4 (early Golgi), Sec7-GFP (late Golgi), or Vps8-GFP (PVE) in a pdr1Δ pdr3Δ or pdr1Δ pdr3Δ vps10-104 background were grown to mid-log phase, treated with SLF for 5 min, fixed, and imaged by confocal microscopy. Shown are representative projected Z-stacks. Arrowheads indicate examples of structures containing detectable cargo, and arrows indicate examples of structures lacking detectable cargo. Scale bar, 2 µm. (B) Quantification from A of the percentages of labeled structures containing detectable cargo. Data were obtained from at least 40 cells per condition. Error bars indicate SEM. (C) Immunoblot of cell-associated and secreted cargo after SLF addition in rich medium. Yeast were grown to mid-log phase in YPD, washed twice with fresh YPD, and treated with SLF. At the indicated time points, cell-associated and secreted fractions were separated by centrifugation, and then the sedimented cells were lysed and proteins from both fractions were precipitated. Samples were subjected to endo H treatment followed by SDS-PAGE and immunoblotting for FKBP. The visualized band corresponds to the upper band in the “+” lane of D. (D) Immunoblot demonstrating hyperglycosylation of the secreted cargo. Proteins were precipitated from rich medium 60 min after SLF addition. Samples were then treated or mock treated with endo H, followed by SDS-PAGE and immunoblotting for FKBP. A high-molecular-weight smear was seen only in the absence of endo H treatment. In the endo H–treated sample, the upper band is the full-length fusion protein, and the lower band is a minor species that was exaggerated by overloading the gel. The lower band might have been generated by Kex2 protease cleavage (Rockwell et al., 2002) of FKBP after the KR dipeptide at positions 17–18. MW, molecular weight marker, with the sizes of the reference proteins shown at the left. (E) Visualization of secreted cargo trapped in the periplasm in minimal medium. Cells expressing the aggregated cargo and the ER marker Erg11-GFP were grown to mid-log phase in NSD buffered at pH 6, then treated with SLF for 30 min and mounted in a flow chamber. Unbuffered NSD (pH ∼4) was flowed over the cells while imaging with a confocal microscope. Shown are projected Z-stacks from Video 2. Scale bar, 2 µm.