Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.
The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.
Krawczyk
Ewa
E
Department of Pathology, Georgetown University Medical School, 3900 Reservoir Rd. NW, Washington, DC 20057, USA.
Suprynowicz
Frank A
FA
Sudarshan
Sawali R
SR
Schlegel
Richard
R
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002584:Cervix Uteri / Q000166:cytology / Q000821:virology; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000821:virology; D005260:Female; D052162:Human papillomavirus 16 / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D006801:Humans; D007425:Intracellular Membranes / Q000821:virology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000086082:Viroporin Proteins
D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins