A different intracellular distribution of a single reporter protein is determined at steady state by KKXX or KDEL retrieval signals.
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.
Lotti
L V
LV
Istituto Nazionale Ricerca sul Cancro di Genova, Sezione di Biotecnologie, Viale Regina Elena 324, 00161, Rome, Italy.
Mottola
G
G
Torrisi
M R
MR
Bonatti
S
S
D017456:Adenovirus Early Proteins / Q000378:metabolism*; D000818:Animals; D016827:CD8 Antigens / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D051381:Rats; D018000:Receptors, Peptide / Q000378:metabolism*; D015398:Signal Transduction
D017456:Adenovirus Early Proteins; D016827:CD8 Antigens; C064459:KDEL receptor; C084004:LMAN1 protein, human; C501458:Lman1 protein, rat; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D018000:Receptors, Peptide