Different fate of a single reporter protein containing KDEL or KKXX targeting signals stably expressed in mammalian cells.
In mammalian cells, resident luminal and type I transmembrane proteins of the endoplasmic reticulum usually contain KDEL and KKXX at the carboxyl terminus. These sequences induce retrieval from compartments located downstream in the secretory pathway. It has been suggested that the retrieval may occur from multiple sites, ranging from the intermediate compartment to the trans-Golgi network. To compare the retrieval of luminal and type I membrane proteins, we have used different forms of a single reporter, the human CD8 glycoprotein, stably expressed in FRT cells. Metabolic labeling and oligosaccharide analysis show that the mechanism based on the KDEL signal is leaky. With time, the KDEL-containing CD8 form reaches the trans/trans-Golgi network compartments, where the protein is terminally glycosylated. At this stage, the retrieval mechanism stops being effective and the protein is consequently secreted. Conversely, the mechanism based on the KKXX signal guarantees that most of the KKXX-containing CD8 form resides in the endoplasmic reticulum, little in the Golgi complex and undetectable levels at the plasma membrane. The O-glycosylation of this protein comprises for the vast majority the sole addition of peptide-bound GalNAc that occurs in an early Golgi compartment.
Martire
G
G
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II," 80131 Naples, Italy.
Mottola
G
G
Pascale
M C
MC
Malagolini
N
N
Turrini
I
I
Serafini-Cessi
F
F
Jackson
M R
MR
Bonatti
S
S
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016827:CD8 Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008322:Mammals; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009842:Oligopeptides; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D015703:Antigens, CD; D016827:CD8 Antigens; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C073762:lysyl-aspartyl-glutamyl-leucine