Abstract
Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.
Highlights
Perturbation of cellular cholesterol homeostasis is seen in cystic fibrosis (CF)2 and other protein folding diseases [1,2,3,4,5]
The global nature of lipid effects observed in CF patients and the relation to protein folding in the endoplasmic reticulum (ER) led us to hypothesize that VAMP-associated proteins (VAPs) might be involved in regulated coupling of protein folding with lipid homeostasis
Cells derived from CF patients or ones that express exogenous ⌬F508-CFTR were previously shown to accumulate abnormal levels of free endosomal cholesterol [2, 6], and we verified these observations in HEK293 cells
Summary
Chemicals, and Antibodies—HeLa, HEK293, and ⌬F508-CFTR stably expressing HEK293 cells and the RNF5/ RMA1 knockdown HeLa cell lines Prywes (Addgene plasmid 11975) and encodes a 3ϫFLAG-tagged human ATF6 protein This plasmid was altered by site-directed mutagenesis using the forward primer 5Ј-CCAGAGGCTTAAAGTCCCTAGTTGAAAGCGAAGAGTTGTCTGTGTGATGATAGTATTGGC-3Ј and reverse primer 5Ј-GCCAATACTATCATCACACAGACAACTCTTCGCTTTCAACTAGGGACTTTAAGCCTCTGG-3Ј to generate a stop codon at amino acid 374 (P374X) and produce the N-terminal nuclear form of ATF6 (3ϫFLAG-nATF6). GST Pulldowns—Both the wild type and 2DK-MSP domains of VAPB were PCR-amplified with the forward primer 5Ј-CATAGGATCCATGGCGAAGGTGGAACAGG-3Ј and the reverse primer 5Ј-GCGCGAATTCCTATTCAAACACACATCTAAGTTTTGAATCC-3Ј to add 5Ј-BamHI and 3Ј-EcoRI sites that were cloned into the pGEX-4T-1 expression vector. GST-VAPB-MSP proteins (250 g for interaction analysis and 50 g for binding competition analysis) were pre-bound to beads, incubated with HeLa cell lysates (prepared in 25 mM Hepes, pH 7.2, 125 mM KOAc, 5 mM MgOAc, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 1% Triton X-100, 1 mM PMSF, 1 mM orthovanadate), collected by centrifugation, washed, and analyzed by Western blots with selective antibodies or mass spectrometry. Quantitative densitometry was conducted using the Quantity One software (Bio-Rad)
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