PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

• Klíčová slova
• Arabidopsis thaliana, Auxin homeostasis and transport, Digitonin PM-permeabilizing protocols, Endoplasmic reticulum, Immunocytochemistry, Membrane topology determination methods, PIN auxin efflux carriers,
• Publikační typ
• časopisecké články MeSH

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.

• MeSH
• alely MeSH
• anemie genetika   MeSH
• biopsie MeSH
• chronická nemoc MeSH
• dánio pruhované embryologie   genetika   MeSH
• dítě MeSH
• dominantní geny MeSH
• dospělí MeSH
• endoplazmatické retikulum metabolismus   MeSH
• exom genetika   MeSH
• fenotyp MeSH
• Golgiho aparát metabolismus   MeSH
• heterozygot * MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA analýza   genetika   MeSH
• missense mutace genetika   MeSH
• mladý dospělý MeSH
• molekulární modely MeSH
• mutace * MeSH
• nemoci ledvin genetika   patologie   MeSH
• neutropenie genetika   MeSH
• novorozenec MeSH
• progrese nemoci MeSH
• rodokmen MeSH
• růstová retardace plodu genetika   MeSH
• sekvence aminokyselin MeSH
• senioři MeSH
• syndrom MeSH
• translokační kanály SEC chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• messenger RNA MeSH
• SEC61A1 protein, human MeSH Prohlížeč
• translokační kanály SEC MeSH