Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease.
Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.
Bracale
Aurora
A
Dipartimento di Chimica Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Spalletti-Cernia
Daniela
D
Mastronicola
Mariarosaria
M
Castaldi
Francesco
F
Mannucci
Roberta
R
Nitsch
Lucio
L
D'Alessio
Giuseppe
G
D016475:3T3 Cells; D000818:Animals; D002417:Cattle; D002461:Cell Line, Transformed; D004251:DNA Transposable Elements; D004722:Endoribonucleases / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D014644:Genetic Variation; D005834:Genital Neoplasms, Male / Q000201:enzymology; D008297:Male; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry; D021382:Protein Sorting Signals; D021122:Protein Subunits; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012661:Semen / Q000201:enzymology; D021601:trans-Golgi Network / Q000201:enzymology
D004251:DNA Transposable Elements; D009363:Neoplasm Proteins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011994:Recombinant Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; D004722:Endoribonucleases; C032523:ribonuclease SPL