Proteins that 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 ER and ultimately cause disease, such as diabetes, liver disease, or cystic fibrosis. The most well characterized pathways for degradation of misfolded proteins in the ER involve retrotranslocation to the cytosol followed by proteasomal degradation. These pathways are categorized as ER associated degradation (ERAD) (Figure 1).
However, some misfolded proteins are poorly recognized by the ERAD system, necessitating their disposal by other pathways that are not well characterized. To learn more about these pathways, we have created misfolded fluorescent protein (FP)-tagged proteins, including a misfolded prion protein (Figures 1 & 2), that accumulate in the ER and are not efficiently degraded by ERAD. We are using a combination of high-resolution live-cell imaging and biochemical techniques to reveal how these model substrates are targeted for degradation. We anticipate that the findings from this study will provide novel insights into the regulation of degradation pathways utilized by disease-related, ER-retained proteins.
Figure 1: As scientists examine a greater variety of ER quality control substrates that differ according to the number of transmembrane domains or post-translational modifications, they discover that the cell has evolved multiple ER-associated degradation (ERAD) pathways to accommodate this diversity. However, degradation pathways of misfolded secretory pathway proteins that are not efficient ERAD substrates are as yet unknown. To reveal novel ER degradation pathways, we study GPI-anchored protein quality control using a quantitatively misfolded variant of YFP-tagged PrP (PrPmisf) as a model.
Figure 2: The major population of PrPmisf is localized in the ER in NRK cells. These cells were co-transfected with a CFP-labeled ER resident protein.