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Methods Enzymol. 2019;619:47-69. doi: 10.1016/bs.mie.2018.12.033. Epub 2019 Feb 8.

Methodologies to monitor protein turnover at the inner nuclear membrane.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States.
2
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States; Department of Cell Biology, Yale School of Medicine, New Haven, CT, United States. Electronic address: christian.schlieker@yale.edu.

Abstract

Lamin B receptor (LBR) is an inner nuclear membrane protein that associates with the nuclear lamina and harbors sterol reductase activity essential for cholesterol biosynthesis. Several LBR mutations implicated in human congenital disorders give rise to C-terminal truncations which render LBR metabolically unstable, resulting in their rapid turnover in the nucleus. These LBR variants serve as model substrates for investigating the poorly understood protein quality control pathways in the mammalian nuclear envelope (NE). Here we describe a split-GFP-based method for tagging these model substrates to enable live cell imaging and flow cytometry for the identification and characterization of NE-resident protein turnover machinery. Furthermore, we describe a facile subcellular fractionation method to isolate a soluble LBR degradation intermediate, allowing the deconvolution of the membrane extraction and proteasomal turnover steps. The combination of imaging-based and biochemical approaches described here facilitates detailed mechanistic studies to dissect protein turnover in the nuclear compartment.

KEYWORDS:

ERAD; Inner nuclear membrane; Lamin B receptor; Nuclear lamina; Nucleus; Proteasome; Ubiquitin; p97; p97 inhibitor

PMID:
30910029
PMCID:
PMC6457266
[Available on 2020-02-08]
DOI:
10.1016/bs.mie.2018.12.033

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