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Description
A mutant of the vacuolar proteinase A (ngPrAdelta295-331) (green) is a substrate of ER quality control (ERQC) mechanisms, as determined by the fact that it is retained in the ER (marked by Kar2; red) when degradation is blocked through deletion mutation of hrd1. S. cerevisiae mutant hrd1::KANMX (deletion mutation of hrd1) bearing ngPRdelta295-331-HA centromeric plasmid were fixed in 3.7% formaldehyde. The cell wall was removed by zymolyase digestion in 1.2M sorbitol and washed resuspended cells were applied to poly L-lysine coated slide, methanol (-20C) and acetone treated, and air dried. Primary antibodies HA.11 mAb (Covance) and polyclonal rabbit anti-Kar2p were applied in 0.05% Tween 20 followed by Alexa Fluor 488 goat anti–mouse and Alexa Fluor 594 goat anti–rabbit (Invitrogen) secondary antibodies. Slides were mounted in PBS, 90% glycerol and DAPI (blue). Cells were visualized using an inverted microscope (LSM 510 META; Carl Zeiss, Inc.) with a Plan Apochromat 100× 1.4 NA Ph3 objective (Carl Zeiss, Inc.) in immersion oil (Immersol 518F; Carl Zeiss, Inc.) at room temperature. Image acquisition was performed using standard photomultiplier tube with LSM 510. Images were archived using LSM 5 Image Examiner (Carl Zeiss, Inc.) and Photoshop (version 7.0; Adobe), and no additional software adjustments were performed on images after acquisition other than cropping. Image corresponds to Fig 1D in J Cell Biol. 2010. 188:707-716. Research Data Curation Program, UC San Diego, La Jolla, 92093-0175 (https://lib.ucsd.edu/rdcp) Kanehara, Kazue; Xie, Wei; Ng, Davis T.W. (2021). CIL:13486, Saccharomyces cerevisiae. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J02F7M2J J Cell Biol. 2010. 188: 707-716. https://www.ncbi.nlm.nih.gov/pubmed/?term=20212318
Type
Dataset
Language
No linguistic content; Not applicable
Subject
Response to unfolded protein ATPase activity Endoplasmic reticulum unfolded protein response SRP-dependent cotranslational protein targeting to membrane, translocation ATP binding Unfolded protein binding ER-associated protein catabolic process Ubiquitin-protein ligase activity Zinc ion binding Nucleus Hrd1p ubiquitin ligase ERAD-L complex Endoplasmic reticulum membrane Endoplasmic reticulum lumen Hrd1p ubiquitin ligase ERAD-M complex Saccharomyces cerevisiae Cell Image Library Group ID: 7401
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