RILP Induces Cholesterol Accumulation in Lysosomes by Inhibiting Endoplasmic Reticulum-Endolysosome Interactions.

Area of Science:

• Cell Biology
• Molecular Biology
• Organelle Biology

Background:

• Endoplasmic reticulum (ER) and endolysosome interactions are crucial for cellular cholesterol homeostasis.
• Vesicle-associated membrane protein-associated protein-OSBP-related protein 1L (VAP-ORP1L) interaction mediates ER-lysosome membrane contact, regulated by Rab7.
• The precise role of Rab7-interacting lysosomal protein (RILP) in ER-lysosome interactions remained unclear.

Purpose of the Study:

• To investigate the role of RILP in ER-lysosome interaction and cholesterol transport.
• To elucidate the mechanism by which RILP affects organelle contact sites.
• To determine the downstream consequences of RILP-mediated interference with ER-endolysosome communication.

Main Methods:

• Immunofluorescence microscopy to visualize organelle positioning and contact sites.
• Protein interaction studies to assess RILP's effect on VAP-ORP1L complex formation.
• Cholesterol level analysis in endolysosomes upon RILP manipulation.
• Autophagy assays to evaluate cellular response to altered cholesterol transport.

Main Results:

• RILP directly interacts with ORP1L, competitively inhibiting VAP-ORP1L contact site formation.
• RILP expression leads to clustering of late endosomes/lysosomes, reducing ER-endolysosome proximity.
• Overexpression of RILP causes cholesterol accumulation in endolysosomes and induces RILP-dependent autophagy.

Conclusions:

• RILP disrupts ER-endolysosome interaction by inhibiting VAP-ORP1L contact.
• This disruption impairs cholesterol flow from endolysosomes to the ER.
• Accumulated endolysosomal cholesterol triggers a feedback loop activating cellular autophagy.