Related Experiment Video
Updated: Jul 16, 2026
Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins
Published on: October 4, 2017
Intracellular pathway of Onconase that enables its delivery to the cytosol
Montserrat Rodríguez1, Gerard Torrent, Montserrat Bosch
1Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Universitat de Girona, Campus de Montilivi s/n E-17071 Girona, Spain.
Abstract:
Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol.
Insights
Onconase, an anti-cancer ribonuclease (RNase), enters cells via clathrin-mediated endocytosis. Its toxicity is enhanced by neutralizing endosomal pH, facilitating cytosolic delivery for targeted cancer cell death.
Area of Science:
- Cell Biology
- Molecular Oncology
- Biochemistry
Background:
- Onconase is a ribonuclease (RNase) selectively toxic to cancer cells.
- Its mechanism of cellular uptake and toxicity has remained largely uncharacterized.
- Previous studies suggested endocytic uptake is crucial for Onconase's cytotoxic effects.
Purpose of the Study:
- To elucidate the specific endocytic pathway utilized by Onconase for cellular entry.
- To identify the intracellular compartments involved in Onconase's cytotoxic mechanism.
- To investigate the role of endosomal pH in Onconase translocation and toxicity.
Main Methods:
- Investigated Onconase internalization using AP-2/clathrin-mediated endocytosis markers.
- Tracked Onconase intracellular trafficking in conjunction with transferrin.
- Utilized tetanus toxin light chain expression to manipulate Onconase concentration in recycling endosomes.
- Performed cell-free translocation assays with purified endosomes and varied endosomal pH.
- Assessed the requirement of ATP hydrolysis for Onconase translocation.
Main Results:
- Onconase internalization occurs via AP-2/clathrin-mediated endocytosis.
- Onconase is routed to the receptor recycling compartment, unlike other RNases.
- Neutralizing endosomal pH significantly enhances Onconase translocation and toxicity (up to 100-fold).
- Onconase translocation from endosomes to the cytosol requires ATP hydrolysis.
Conclusions:
- Onconase employs a unique clathrin-dependent endocytic pathway for cellular entry.
- Recycling endosomes serve as a critical compartment for Onconase delivery to the cytosol.
- Endosomal pH neutralization is a key factor for efficient Onconase translocation and enhanced anti-cancer activity.
Related Concept Videos
01:34Protein Transport into the Inner Mitochondrial Membrane
Transport of mitochondrial precursors across the TIM23 channel is driven by...
01:36Delivery Pathways to the Lysosome
Endocytosis
In endocytosis, the cell membrane takes up macromolecules and particles from the surrounding medium. Clathrin-mediated...
01:19Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:28Protein Translocation Machinery on the ER Membrane
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.
01:17The Extrinsic Apoptotic Pathway
01:45ER Retrieval Pathway
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms...