A new mitofusin topology places the redox-regulated C terminus in the mitochondrial intermembrane space

Mitochondrial fusion occurs in many eukaryotes, including animals, plants, and fungi. It is essential for cellular homeostasis, and yet the underlying mechanisms remain elusive. Comparative analyses and phylogenetic reconstructions revealed that fungal Fzo1 and animal Mitofusin proteins are highly diverged from one another and lack strong sequence similarity. Bioinformatic analysis showed that fungal Fzo1 proteins exhibit two predicted transmembrane domains, whereas metazoan Mitofusins contain only a single transmembrane domain. This prediction contradicts the current models, suggesting that both animal and fungal proteins share one topology. This newly predicted topology of Mfn1 and Mfn2 was demonstrated biochemically, confirming that the C-terminal, redox-sensitive cysteine residues reside within the intermembrane space (IMS). Functional experiments established that redox-mediated disulfide modifications within the IMS domain are key modulators of reversible Mfn oligomerization that drives fusion. Together, these results lead to a revised understanding of Mfns as single-spanning outer membrane proteins with an Nout–Cin orientation, providing functional insight into the IMS contribution to redox-regulated fusion events.

Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK) were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

Identification of Genes Potentially Involved in the Biocontrol Activity of Pseudozyma flocculosa

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100×) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.

Swapping the Gene-Specific and Regional Silencing Specificities of the Hst1 and Sir2 Histone Deacetylases

ABSTRACT Sir2 and Hst1 are NAD+-dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved N-terminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.

Eng1p, an Endo-1,3-β-Glucanase Localized at the Daughter Side of the Septum, Is Involved in Cell Separation in Saccharomyces cerevisiae

ABSTRACT ENG1 (YNR067c), a gene encoding a new endo-1,3-β-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-β-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the Saccharomyces cerevisiae ACF2 gene, involved in actin assembly “in vitro,” and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-β linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds. ENG1 transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G1 transition and is dependent on the transcription factor Ace2p. Interestingly, eng1 deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.

Characterization of U2AF6, a Splicing Factor Related to U2AF35

ABSTRACT The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. U2AF35 has multiple functions in pre-mRNA splicing. First, U2AF35 has been shown to function by directly interacting with the AG at the 3′ splice site. Second, U2AF35 is thought to play a role in the recruitment of U2AF65 by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF35 and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF35, designated U2AF26. The N-terminal 187 amino acids of U2AF35 and U2AF26 are nearly identical. However, the C-terminal domain of U2AF26 lacks many characteristics of the U2AF35 RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF26 can associate with U2AF65 and can functionally substitute for U2AF35 in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF26 functions by enhancing the binding of U2AF65 to weak 3′ splice sites. These studies identify U2AF26 as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF35, U2AF26 may play a role in regulating alternative splicing.

Complete nucleotide sequence, genomic organization and phylogenetic analysis of a novel genital human papillomavirus type, HLT7474-S

A novel human papillomavirus (HPV) type, HLT7474-S, was isolated from a cervical scraping of a female sex worker with a wart virus infection. The complete DNA sequence of 7812 bp was derived from four overlapping PCR products and authenticated by RFLP analysis. The L1 gene exhibited 78% identity to those of its most closely related known HPV types in group A7, comprising HPV types 18, 39, 45, 59, 68 and 70. The genomic organization and phylogenetic analysis of HLT7474-S and group A7 HPVs reiterated their relatedness. Of significance were the strong sequence similarity, phylogenetic relationship and conservation of critical motifs between the transforming E6 and E7 of HLT7474-S and E6 of HPV-18 and E7 of HPV-59, respectively. These features clearly suggest that HLT7474-S is a high-risk genital HPV isolate, closely related to HPV-18 and other members of the A7 group of genital HPVs.

Transposon-Disruption of a Maize Nuclear Gene, tha1, Encoding a Chloroplast SecA Homologue: In Vivo Role of cp-SecA in Thylakoid Protein Targeting

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

The characterization of a cyclophilin-type peptidyl prolyl cis-trans-isomerase from the endoplasmic-reticulum lumen

A luminally located peptidyl prolyl cis-trans-isomerase (PPI) has been purified from bovine liver microsomes. It has a molecular mass of 20.6 kDa, and N-terminal sequencing demonstrates strong sequence similarity to the sequences of the cyclophilin B family. The enzyme catalyses the isomerization of the standard proline-containing peptide N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, as well as the refolding of RNAase T1. Kinetic properties, substrate-specificity data and inhibition by cyclosporin A indicate that it is a cyclophilin-type PPI, consistent with the amino-acid-sequence results.

The human carbonic anhydrase I gene has two promoters with different tissue specificities

Primer extension and S1 nuclease analysis of human carbonic anhydrase I (HCA1) mRNA transcripts in erythroid and non-erythroid tissues show that the HCA1 gene has two promoters with different tissue specificities, separated by 36 kb of DNA. The promoter of the HCA1 gene which is active in non-erythroid tissue shows strong sequence similarity with a similar region in the mouse CA1 gene, but has two equally used transcription start sites.