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Different endoplasmic reticulum trafficking and processing pathways for calsequestrin (CSQ) and epitope-tagged CSQ
Timothy D Houle1, Michal L Ram, Walter J McMurray
1Wayne State University, Elliman Building, Room 1107, 421 East Canfield Avenue, Detroit, MI 48201, USA.
Experimental Cell Research
|October 19, 2006
Summary
Cardiac calsequestrin (CSQ) protein concentrates in the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). CSQ polymerization appears to be a key mechanism for its ER/SR retention.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- Cardiac calsequestrin (CSQ) is found in sarcoplasmic reticulum (SR) terminal cisternae, compartments of unclear origin.
- Investigating CSQ trafficking within standard ER compartments is crucial for understanding its cellular localization.
Purpose of the Study:
- To investigate the trafficking and localization of cardiac calsequestrin (CSQ) within standard ER compartments.
- To determine the molecular structure and post-translational modifications of CSQ in nonmuscle cells.
Main Methods:
- Expression of CSQ in nonmuscle cell lines.
- Immunofluorescence microscopy for protein localization.
- Mass spectrometry for molecular structure analysis.
- Analysis of glycosylation and phosphorylation patterns.
Main Results:
- CSQ is a highly phosphorylated protein with ER-retention-predictive glycan structures (Man9,8GlcNAc2).
- Immunostaining localized CSQ to polymeric ER cisternae.
- Disruption of the secretory pathway altered CSQ glycosylation and phosphorylation.
- Epitope-tagged CSQ exhibited reduced ER staining and altered post-translational modifications compared to wild-type CSQ.
Conclusions:
- CSQ polymerization is identified as a potential mechanism for ER/SR retention.
- Loss of ER retention is linked to epitope tags, thapsigargin treatment, changes in protein structure, or Ca(2+) levels.
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