Alternative Splicing Gives Rise to Different Isoforms of the Neurospora crassa Tob55 Protein That Vary in Their Ability to Insert β-Barrel Proteins Into the Outer Mitochondrial Membrane

Abstract

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import b-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by $4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly b-barrel proteins.

Analysis of Mutations in Neurospora crassa ERMES Components Reveals Specific Functions Related to β-Barrel Protein Assembly and Maintenance of Mitochondrial Morphology

“…We previously suggested that N. crassa Mdm10 has a general effect on TOB complex function [26]. Interestingly, our model fits with certain aspects of the one described above since N. crassa strains lacking Mdm10 do have a porin assembly defect [26] and a stable intermediate for porin assembly can be detected in N. crassa [36]. A third model suggests that the role of Mdm10 is to assist the TOB complex with the assembly of Tom22 [29], [30].…”

“…Strains were grown at 30°C unless otherwise noted. General handling procedures for N. crassa [35] and growth tests using sorbose containing medium to force colonial growth were as described previously [36]. The deletion strains used in this study ( Δmmm1 , Δmmm2 , and Δgem1) were developed by the N. crassa gene knockout project [37].…”

“…Blue native gel electrophoresis (BNGE) [45], [46], Western blotting [47], import and assembly of proteins into isolated mitochondria that were not sucrose gradient purified (crude mitochondria) [48], alkaline extraction [26], [27], isolation of outer membrane vesicles [49], and transformation of N. crassa [36] were performed as described previously. Mitochondrial proteins were analyzed by SDS-PAGE as previously described [50].…”

Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae

“…Since then, many cases of alternative splicing have been observed. As examples, alternative splicing was observed in the Chlorella sorokiniana nadp-gdh gene (Miller et al, 1998); S. cerevisiae YKL186C/MTR2 and YML034W genes (Davis et al, 2000); Pneumocystis carinii impdh gene (Ye et al, 2001); Mucor circinelloides rce1 gene (Baba et al, 2005); N. crassa tob55 gene (Hoppins et al, 2007) and N. crassa hex-1 gene (Leal et al, 2009). In all the cited works, intron retention rather than the formation of exon cassettes seems to be the prevalent alternative splicing event in fungi (McGuire et al, 2008).…”

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

“…The first heterokaryon (RIP40het) was isolated (19) using the technique of sheltered RIP (repeat-induced point mutation). The second (Tom40 KO-5) was developed by the method of sheltered disruption in heterokaryon HP1 as described previously for mutants of Tob55 (42). The nuclei of the heterokaryons can be differentiated and selected using differing auxotrophic and drug-resistance markers.…”