Abstract: FR-PO383
APOL1 Alternative Splice Isoforms Localize to the Endoplasmic Reticulum with Distinct Topologies and Do Not Associate with Endosomes
Session Information
- CKD: Mechanisms - II
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Scales, Suzie J., Genentech, South San Francisco, California, United States
- Oltrabella, Francesca, Genentech, South San Francisco, California, United States
- Gupta, Nidhi, Genentech, South San Francisco, California, United States
Background
Two risk variants of the APOL1 gene, G1 and G2, are associated with chronic kidney disease in African Americans. APOL1 risk variants have been proposed to cause podocyte toxicity by a variety of pathways, including inhibition of endosomal maturation via direct binding to cytoplasmic VAMP8 on endosomes. APOL1 G0 (wild type), G1 and G2 variants all localize to the endoplasmic reticulum (ER) and the cell surface of podocytes, suggesting that direct interaction with VAMP8 is unlikely. However, those studies utilized isoform APOL1.vA, whereas three minor splice isoforms (vB1, vB3 and vC) exist, with different N-termini and so may be not be secreted. While transient transfection of all four isoforms leads to their apparent secretion, this may be an overexpression artifact. We therefore aimed to determine the localization of APOL1 isoforms stably expressed in podocytes.
Methods
We generated inducible APOL1 stable podocytes and determined the localization of each isoform by immunofluorescence and flow cytometry. We assessed APOL1 isoform levels in immortalized human podocytes by qRT-PCR, immunofluorescence and western blotting.
Results
By immunofluorescence with Triton X-100 permeabilization, all four isoforms localized to the ER, but selective permeabilization of the plasma membrane with digitonin revealed topological differences: isoforms vA and vB1 are inside the ER lumen, while vB3 and most of vC are on the outer face of the ER, like APOL2. Consequently, only vA and vB1 are appreciably detected on the cell surface by flow cytometry, indicating those two isoforms are secretory, while vB3 and vC are cytoplasmic. Wild type podocytes express the three splice isoforms at much lower levels than vA by RT-PCR, but APOL1 mRNA and protein levels are notoriously poorly correlated. Western blotting of endogenous podocyte APOL1 with sensitive in-house APOL1-specific antibodies revealed the major band was vA, but a minor upper band became detectable upon gamma interferon stimulation. This could represent vB3 or a post-translational modification.
Conclusion
APOL1 isoforms vA and vB1 are secretory, whereas vB3 and most of vC are cytoplasmic, but on the outer ER membrane, not endosomal. APOL1 is therefore unlikely to directly interact with VAMP8 to interfere with endosomal trafficking.
Funding
- Commercial Support –