TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employed a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. Over 300 proteins were identified, forming different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown solid inclusion at the ER exit sites (ERES). TDP43-ERES coaggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delayed ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients. ### Competing Interest Statement The authors have declared no competing interest. * 3D : 3 dimension aa : Amino acid AF : Alexa Fluor AGC : Automatic gain control ALS : Amyotrophic Lateral Sclerosis Baf : Bafilomycin A1 BCA : Bicinchoninic acid BDNF : Brain Derived Neurotrophic Factor C9 : C9ORF72 CAA : 2-chloroacetamide ChAT : Choline acetyltransferase CHX : Cycloheximide Coagg : Coaggregate COPII : Coat Protein Complex II CRT : Calreticulin DMEM : Dulbecco’s Modified Eagle Medium DNA : Deoxyribonucleic acid DsiRNA : Dicer-substrate siRNA DWB : Double Walker B EGFP : Enhanced green fluorescent protein ER : Endoplasmic reticulum ERES : ER-exit site FACS : Fluorescence-activated cell sorting FBS : Fetal bovine serum FDR : False discovery rate FISH : Fluorescence in situ hybridization FP : Fluorescent protein FRAP : Fluorescence recovery after photobleaching FUS : Fused in sarcoma GDNF : Glial cell line-derived neurotrophic factor GM130 : Golgi matrix protein 130 kDa Halo : HaloTag HCD : Higher-energy collision dissociation HEK293T : Human embryonic kidney 293T hESC : Human embryonic stem cell Hex : 1,6-Hexanediol hiPSC : Human induced pluripotent stem cell hnRNP A1 : Heterogeneous nuclear ribonucleoprotein A1 Hsp : Heat-shock protein Htt : Huntingtin IDR : Intrinsically disordered region IF : Immunofluorescence iMN : Induced MN INS-1 : (Rat) Insulinoma-1 IP : Immunoprecipitation IPOD : Insoluble protein deposit IT : Injection time JF : Janelia Fluor JUNQ : Juxta-nuclear quality control compartment mEGFP : Monomeric EGFP mNG : Monomeric NeonGreen NeuN : Neuronal nuclei antigen G3BP1 : Ras GTPase-activating protein-binding protein 1 LC : Liquid chromatography LLPS : Liquid-liquid phase separation ManII : Mannosidase II MN : Motor neuron MS : Mass-spectrometry MTOC : Microtubule organization center NBD : Nucleotide-binding domain NEAA : Non-essential amino acids NFM : Neurofilament medium polypeptide protein NM : Neural media NIM : Neural Induction Media NLS : Nuclear localization signal NTD : N-terminal domain PBS : Phosphate buffered saline PBST : PBS + 0.1% [v/v] Tween 20 PCR : Polymerase chain reaction PD : Parkinson’s Disease PFA : Paraformaldehyde PIC : Protease inhibitor cocktail PMSF : Phenylmethylsulfonyl fluoride PolyA : Poly-adenine PS-1 : Presenilin-1 RA : Retinoic acid RBFOX3 : RNA binding protein fox-1 homolog 3 RIPA : Radioimmunoprecipitation assay RNA : Ribonucleic acid RNAi : RNA interference RPE-1 : Retinal pigment epithelial-1 RRM : RNA-recognition motif RT : Room temperature RUSH : Retention Using Selective Hooks sALS : sporadic ALS SB : Sec body SBP : Streptavidin-binding protein SDS : Sodium dodecyl sulfate SDS-PAGE : SDS-polyacrylamide gel electrophoresis SG : Stress granule SHH : Sonic Hedgehog SIM : Structured Illumination Microscopy siRNA : Small interfering RNA SOD1 : Superoxide dismutase 1 SR : Super-resolution ss : (Secretory) signal sequence SSC : Saline-sodium citrate TCEP : tris(2-carboxyethyl)phosphine TDP43 : TAR DNA-binding protein 43 kDa TEAB : Triethylammonium bicarbonate TFA : Trifluoroacetic acid TNFα : Tissue necrosis factor α tRNA : transfer RNA VC : Vitamin C (L-ascorbic acid) WB : Western blot WT : Wild-type