Regulatory Implications of Non-Trivial Splicing: Isoform 3 of Rab1A Shows Enhanced Basal Activity and Is Not Controlled by Accessory Proteins. Issue 8 (24th April 2016)

• Record Type: Journal Article
• Authors: Schöppner, Patricia; Csaba, Gergely; Braun, Tatjana; Daake, Marina; Richter, Bettina; Lange, Oliver F.; Zacharias, Martin; Zimmer, Ralf; Haslbeck, Martin
• Is Part Of: Journal of molecular biology. Volume 428:Issue 8(2016:Apr. 15)
• Page Start: 1544
• Page End: 1557
• Publication Date: 2016-04-24
• Subjects: non-trivial splicing -- Rab1A -- protein folding -- protein secretion; nt-svRab1A non-trivial splice variant, Rab1A isoform 3 -- REP Rab escort protein -- GEFs guanine nucleotide exchange factors -- GAPs GTPase activating proteins -- NGS next-generation sequencing -- SEC size exclusion chromatography -- TSAs thermal shift assays -- MANT methylanthraniloyl -- RFI relative fluorescence intensity -- Co-IP co-immunoprecipitation; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.02.028
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Alternative splicing often affects structured and highly conserved regions of proteins, generating so called non-trivial splicing variants of unknown structure and cellular function. The human small G-protein Rab1A is involved in the regulation of the vesicle transfer from the ER to Golgi. A conserved non-trivial splice variant lacks nearly 40% of the sequence of the native Rab1A, including most of the regulatory interaction sites. We show that this variant of Rab1A represents a stable and folded protein, which is still able to bind nucleotides and co-localizes with membranes. Nevertheless, it should be mentioned that compared to other wild-typeRabGTPases, the measured nucleotide binding affinities are dramatically reduced in the variant studied. Furthermore, the Rab1A variant forms hetero-dimers with wild-type Rab1A and its presence in the cell enhances the efficiency of alkaline phosphatase secretion. However, this variant shows no specificity for GXP nucleotides, a constantly enhanced GTP hydrolysis activity and is no longer controlled by GEF or GAP proteins, indicating a new regulatory mechanism for the Rab1A cycle via alternative non-trivial splicing. Graphical abstract: Highlights: Isoform 3 of Rab1A, a non-trivial splice variant is evolutionary conserved Nt-svRab1A is a nucleotide binding protein, not controlled by regulatory proteins Nt-svRab1A interacts with membranes and forms a hetero-dimer with wtRab1A Nt-svRab1A enhances protein secretion in the cell Non-trivial splicing is an alternative regulatory mechanism for the Rab1A cycle