FRET-assisted determination of CLN3 membrane topology

  • Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, which encodes for a putative lysosomal transmembrane protein with thus far undescribed structure and function. Here we investigate the membrane topology of human CLN3 protein with a combination of advanced molecular cloning, spectroscopy, and in silico computation. Using the transposomics cloning method we first created a library of human CLN3 cDNA clones either with a randomly inserted eGFP, a myc-tag, or both. The functionality of the clones was evaluated by assessing their ability to revert a previously reported lysosomal phenotype in immortalized cerebellar granular cells derived from Cln3Δex7/8 mice (CbCln3Δex7/8). The double-tagged clones were expressed in HeLa cells, and FRET was measured between the donor eGFP and an acceptor DyLight547 coupled to a monoclonal α-myc antibody to assess their relative membrane orientation. The data were used together with previously reported experimental data to compile a constrained membrane topology model for hCLN3 using TOPCONS consensus membrane prediction algorithm. Our model with six transmembrane domains and cytosolic N- and C-termini largely agrees with those previously suggested but differs in terms of the transmembrane domain positions as well as in the size of the luminal loops. This finding improves understanding the function of the native hCLN3 protein.

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Author:Ewa Ratajczak, Anton Petcherski, Juliana Ramos-Moreno, Mika O. Ruonala
Pubmed Id:
Parent Title (English):PLoS One
Place of publication:Lawrence, Kan.
Document Type:Article
Date of Publication (online):2014/07/22
Date of first Publication:2014/07/22
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2014/08/11
Page Number:10
Copyright: © 2014 Ratajczak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Institutes:Wissenschaftliche Zentren und koordinierte Programme / Center for Membrane Proteomics (CMP)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlung Biologie / Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 4.0