Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

Peroxisomes are juxtaposed to strategic sites on mitochondria.

Peroxisomes are ubiquitous and dynamic organelles that house many important pathways of cellular metabolism. In recent years it has been demonstrated that mitochondria are tightly connected with peroxisomes and are defective in several peroxisomal diseases. Indeed, these two organelles share metabolic routes as well as resident proteins and, at least in mammals, are connected via a vesicular transport pathway. However the exact extent of cross-talk between peroxisomes and mitochondria remains unclear. Here we used a combination of high throughput genetic manipulations of yeast libraries alongside high content screens to systematically unravel proteins that affect the transport of peroxisomal proteins and peroxisome biogenesis. Follow up work on the effector proteins that were identified revealed that peroxisomes are not randomly distributed in cells but are rather localized to specific mitochondrial subdomains such as mitochondria-ER junctions and sites of acetyl-CoA synthesis. Our approach highlights the intricate geography of the cell and suggests an additional layer of organization as a possible way to enable efficient metabolism. Our findings pave the way for further studying the machinery aligning mitochondria and peroxisomes, the role of the juxtaposition, as well as its regulation during various metabolic conditions. More broadly, the approaches used here can be easily applied to study any organelle of choice, facilitating the discovery of new aspects in cell biology.

The role of the endoplasmic reticulum in peroxisome biogenesis.

Peroxisomes are essential cellular organelles involved in lipid metabolism. Patients affected by severe peroxisome biogenesis disorders rarely survive their first year. Genetic screens in several model organisms have identified more than 30 PEX genes that are required for the formation of functional peroxisomes. Despite significant work on the PEX genes, the biogenic origin of peroxisomes remains controversial. For at least two decades, the prevailing model postulated that peroxisomes propagate by growth and fission of preexisting peroxisomes. In this review, we focus on the recent evidence supporting a new, semiautonomous model of peroxisomal biogenesis. According to this model, peroxisomal membrane proteins (PMPs) traffic from the endoplasmic reticulum (ER) to the peroxisome by a vesicular budding, targeting, and fusion process while peroxisomal matrix proteins are imported into the organelle by an autonomous, posttranslational mechanism. We highlight the contradictory conclusions reached to answer the question of how PMPs are inserted into the ER. We then review what we know and what still remains to be elucidated about the mechanism of PMP exit from the ER and the contribution of preperoxisomal vesicles to mature peroxisomes. Finally, we discuss discrepancies in our understanding of de novo peroxisome biogenesis in wild-type cells. We anticipate that resolving these key issues will lead to a more complete picture of peroxisome biogenesis.

Polymorphisms in multiple genes contribute to the spontaneous mitochondrial genome instability of Saccharomyces cerevisiae S288C strains.

The mitochondrial genome (mtDNA) is required for normal cellular function; inherited and somatic mutations in mtDNA lead to a variety of diseases. Saccharomyces cerevisiae has served as a model to study mtDNA integrity, in part because it can survive without mtDNA. A measure of defective mtDNA in S. cerevisiae is the formation of petite colonies. The frequency at which spontaneous petite colonies arise varies by approximately 100-fold between laboratory and natural isolate strains. To determine the genetic basis of this difference, we applied quantitative trait locus (QTL) mapping to two strains at the opposite extremes of the phenotypic spectrum: the widely studied laboratory strain S288C and the vineyard isolate RM11-1a. Four main genetic determinants explained the phenotypic difference. Alleles of SAL1, CAT5, and MIP1 contributed to the high petite frequency of S288C and its derivatives by increasing the formation of petite colonies. By contrast, the S288C allele of MKT1 reduced the formation of petite colonies and compromised the growth of petite cells. The former three alleles were found in the EM93 strain, the founder that contributed approximately 88% of the S288C genome. Nearly all of the phenotypic difference between S288C and RM11-1a was reconstituted by introducing the common alleles of these four genes into the S288C background. In addition to the nuclear gene contribution, the source of the mtDNA influenced its stability. These results demonstrate that a few rare genetic variants with individually small effects can have a profound phenotypic effect in combination. Moreover, the polymorphisms identified in this study open new lines of investigation into mtDNA maintenance.