Abstract
To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.
Highlights
Many resident proteins of the ER bear short carboxyl-terminal sequences, KKXX in type I membrane proteins and KDEL in luminal proteins [1, 2]
To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells
The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex
Summary
Antibodies—The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), from Ortho (Raritan, NJ), mouse mAb N1 (anti-CD8 protein) [19], rabbit polyclonal anti-CD8 P. Paccaud, University of Geneva, Switzerland), rabbit polyclonal anti--COP University of Geneva, Switzerland), mouse mAb anti-KDELr National University of Singapore, Singapore), rabbit polyclonal anti-calnexin Ultrathin sections of LR White- or Lowicryl K4Membedded cells were collected on nickel grids and immunolabeled with anti-CD8 followed by protein A-gold (18 nm; 24). The sections were first incubated with anti-CD8 antibody followed by 10 nm of protein A-gold and treated with the second set of antibodies followed by 18 nm of protein-A gold. Thin sections incubated with biotinylated-HPL and anti-biotin colloidal gold were successively immunolabeled with anti-CD8 followed by protein A-colloidal gold (18 nm). Quantitative evaluation of immunolabeling was performed as described previously [25]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
