RESUMEN

Bacteriophages constitute a ubiquitous threat to bacteria, and bacteria have evolved numerous anti-phage defense systems to protect themselves. These systems include well-studied phenomena such as restriction endonucleases and CRISPR, while emerging studies have identified many new anti-phage defense systems whose mechanisms are unknown or poorly understood. Some of these systems involve overcoming lysogenization defect (OLD) nucleases, a family of proteins comprising an ABC ATPase domain linked to a Toprim nuclease domain. Despite being discovered over 50 years ago, OLD nuclease function remained mysterious until recent biochemical, structural, and bioinformatic studies revealed that OLD nucleases protect bacteria by functioning in diverse anti-phage defense systems including the Gabija system and retrons. In this review we will highlight recent discoveries in OLD protein function and their involvement in multiple discrete anti-phage defense systems.

RESUMEN

Borate transporters are membrane transport proteins that regulate intracellular borate levels. In plants, borate is a micronutrient essential for growth but is toxic in excess, while in yeast, borate is unnecessary for growth and borate export confers tolerance. Borate transporters share structural homology with human bicarbonate transporters in the SLC4 family despite low sequence identity and differences in transported solutes. Here, we characterize the S. cerevisiae borate transporter Bor1p and examine whether key biochemical features of SLC4 transporters extend to borate transporters. We show that borate transporters and SLC4 transporters share multiple properties, including lipid-promoted dimerization, sensitivity to stilbene disulfonate-derived inhibitors, and a requirement for an acidic residue at the solute binding site. We also identify several amino acids critical for Bor1p function and show that disease-causing mutations in human SLC4A1 will eliminate in vivo function when their homologous mutations are introduced in Bor1p. Our data help elucidate mechanistic features of Bor1p and reveal significant functional properties shared between borate transporters and SLC4 transporters.

RESUMEN

Borate is an essential micronutrient in plants regulated by borate transporters, which also protect both yeast and plants from toxically high levels of borate and share homology with the human SLC4 transporters. SLC4A11 is linked to congenital hereditary endothelial dystrophy and was initially reported to transport borate before subsequent studies rebutted this conclusion. To better understand the transport activities of purported borate transporters, we tested the ability of SLC4A11 and eleven borate transporters from A. thaliana and O. sativa to complement a BOR1 deletion in S. cerevisiae . We show that AtBOR4 , AtBOR5 , AtBOR7 , OsBOR2 , and OsBOR3 can each complement ScBOR1 , while the rest of the transporters tested do not rescue growth. Additionally, quantification of intracellular borate content demonstrates that SLC4A11 does not export borate in yeast, supporting studies that its transported substrate is not borate.

RESUMEN

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

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RESUMEN

Boron is essential for plant growth because of its incorporation into plant cell walls; however, in excess it is toxic to plants. Boron transport and homeostasis in plants is regulated in part by the borate efflux transporter Bor1, a member of the solute carrier (SLC) 4 transporter family with homology to the human bicarbonate transporter Band 3. Here, we present the 4.1-Å resolution crystal structure of Arabidopsis thaliana Bor1. The structure displays a dimeric architecture in which dimerization is mediated by centralized Gate domains. Comparisons with a structure of Band 3 in an outward-open state reveal that the Core domains of Bor1 have rotated inwards to achieve an occluded state. Further structural comparisons with UapA, a xanthine transporter from the nucleobase-ascorbate transporter family, show that the downward pivoting of the Core domains relative to the Gate domains may access an inward-open state. These results suggest that the SLC4, SLC26, and nucleobase-ascorbate transporter families all share an elevator transport mechanism in which alternating access is provided by Core domains that carry substrates across a membrane.

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