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Structural Organization of the Retriever-CCC endosomal recycling complex

ABSTRACT: Protein-protein interaction analysis in Huh7 cells expressing specific VPS35L mutations. LUM2_1066594: EV_rep1 LUM2_1066595: WT_rep1 LUM2_1066596: W6D_rep1 LUM2_1066597: S829E_rep1 LUM2_1066598: G902E_rep1 LUM2_1066599: G325E_rep1 LUM2_1068615: EV_rep2 LUM2_1068616: WT_rep2 LUM2_1068617: W6D_rep2 LUM2_1068618: S829E_rep2 LUM2_1068619: G902E_rep2 LUM2_1068620: G325E_rep2 LUM2_1070655: EV_rep3 LUM2_1070656: WT_rep3 LUM2_1070657: W6D_rep3 LUM2_1070658: S829E_rep3 LUM2_1070659: G902E_rep3 LUM2_1070660: G325E_rep3

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ezra Burstein

PROVIDER: MSV000092102 | MassIVE | Mon Jun 05 08:40:00 BST 2023

REPOSITORIES: MassIVE

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Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain. Three male S control and 3 male congenic S.LEW(10)x12x2x3x5 rats born on the same day were selected, weaned at 30 days of age, and caged with 1 congenic and 1 S rat per cage. They were raised on a low-salt (0.3%) diet (Harlan Teklad diet TD 7034; Harlan–Sprague-Dawley) and sacrificed at 53 days of age and total RNAs were isolated from the kidney. The isolated RNA from each animal was used for the cRNA preparation. cRNA was prepared and fragmented as suggested by Affymetrix technical manual, and simultaneously hybridized (15 µg adjusted cRNA for each chip) to Rat Expression Array 230 2.0 (3' IVT Expression Analysis). Statistical analyses of the microarray data were performed with BH adjustment using R statistical package (version 2.8.1).

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Structural Organization of the Retriever-CCC endosomal recycling complex

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