Selective Degradation of ER-retained Misfolded Proteins
Prasanna Satpute-Krishnan
Proteins which are destined for the plasma membrane or for secretion are synthesized, folded to their proper form, and processed at the endoplasmic reticulum (ER). Under a variety of conditions, proteins become terminally misfolded in the ER and are targeted for degradation by quality control pathways. When this process is impaired, misfolded proteins accumulate in the endoplasmic reticulum (ER) and ultimately cause disease, such as diabetes, liver disease, or cystic fibrosis.
The most well characterized pathway for degradation of misfolded proteins in the ER is ER associated degradation (ERAD), which involves retrotranslocation of the protein to the cytosol and degradation by the proteasome. However, some misfolded proteins are poorly recognized by the ERAD system, necessitating their disposal by other pathway(s) that are not well characterized. To learn more about these pathways, we have created a fluorescent protein (FP)-tagged misfolding protein that is localized in the ER and does not undergo ERAD. We are using high resolution, time-lapse live cell imaging, photoactivation and photobleaching techniques in cells expressing different fluorescent organelle markers to discern the fate of ER-retained misfolding protein. We plan to use both biochemical and microscopy techniques to examine the factors involved in targeting ER-retained misfolded protein for degradation, as well as to look at how the different degradation pathways interact. The findings from this study may provide novel insights into the regulation of degradation pathways utilized by disease-related, ER-retained proteins.
Figure: Shown here (left) is an NRK cell co-expressing CD3 delta-CFP (red) which is primarily degraded by ERAD and a model protein for the study of ERAD, along with a YFP-tagged misfolding variant of the prion protein (green) which does not undergo ERAD. A portion of the image which is outlined by the yellow box has been shown with the separate channels (right). The misfolding variant is segregated from other ER-resident proteins and routed into vesicular membrane-bound compartments, some of which are colocalized with the autophagy marker LC3 and/or the lysosomal marker LAMP1 (data not shown here), suggesting that it undergoes autophagy.
