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The E3-6.7K protein of adenovirus is an Asn-linked integral membrane glycoprotein localized in the endoplasmic
1Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, Missouri 63104.
Abstract:
We have previously shown that the early region E3 of adenovirus type 2 encodes a 6700 MW (6.7K) protein. This protein is immunoprecipitated from infected cells as two series of bands, a doublet at 7-8K and a doublet or triplet at 15-16K. The predicted amino acid sequence of the 6.7K indicates that there are three potential Asn-linked glycosylation sites near the N-terminus of the protein. Studies done using tunicamycin, endoglycosaminidase H (endo H), and endo F demonstrate that 6.7K has exclusively high mannose oligosaccharides and that only one of the three potential glycosylation sites of 6.7K is glycosylated. [3H]-Mannose labeling confirmed that the upper species of 6.7K were glycosylated. Since the oligosaccharides are not processed from high mannose to the complex type, this implies that the 6.7K protein is retained in the endoplasmic reticulum (ER). This was confirmed with immunofluorescence. Based on the predicted amino acid sequence, 6.7K does not have a classical N-terminus signal sequence, but it does have a 22 amino acid hydrophobic domain, located at amino acids 15-36, that could function as a signal-anchor domain. We demonstrate that the 6.7K protein is an integral membrane ER protein. Considering that all of the potential Asn-glycosylation sites are near the N-terminus of 6.7K, it must be oriented in the membrane with its N-terminus in the lumen of the ER and its C-terminus in the cytoplasm. Pulse-chase studies performed to examine the temporal appearance of the different 6.7K moieties suggests that this protein may translocate into the ER membrane post-translationally, or may be glycosylated post-translationally.
Insights
Adenovirus E3 6.7K protein is an integral endoplasmic reticulum membrane protein. It is retained in the ER due to high mannose oligosaccharides, with its N-terminus lumenal and C-terminus cytoplasmic.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Adenovirus early region E3 encodes a 6.7K protein.
- This protein exists as multiple glycosylated forms in infected cells.
Purpose of the Study:
- To characterize the adenovirus type 2 E3 6.7K protein.
- To determine its cellular localization and membrane orientation.
Main Methods:
- Immunoprecipitation and glycosidase treatment (tunicamycin, endo H, endo F).
- Radioactive labeling ([3H]-Mannose).
- Immunofluorescence and pulse-chase studies.
Main Results:
- The 6.7K protein possesses high mannose oligosaccharides, indicating ER retention.
- It contains a hydrophobic domain functioning as a signal-anchor.
- The protein is an integral membrane protein with N-terminus in the ER lumen and C-terminus in the cytoplasm.
Conclusions:
- Adenovirus E3 6.7K protein is an integral ER membrane protein.
- Its N-terminal glycosylation and ER retention are key features.
- Post-translational translocation or glycosylation into the ER membrane is suggested.