The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in β-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.

Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which DeathAssociated Protein Kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thuspromotes Beclin 1 dissociation from Bcl-XL and autophagy induction.1 Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, inline with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-XL proteins. Our results reveal a new phosphorylation-basedmechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.

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.

To optimally perform the diversity of metabolic functions that occur within peroxisomes, cells must dynamically regulate peroxisome size, number and content in response to the cell state and the environment. Except for transcriptional regulation little is known about the mechanisms used to perform this complicated feat. Focusing on the yeast Saccharomyces cerevisiae, we used complementary high-content screens to follow changes in localization of most proteins during growth in oleate. We found extensive changes in cellular architecture and identified several proteins that colocalized with peroxisomes that had not previously been considered peroxisomal proteins. One of the newly identified peroxisomal proteins, Ymr018w, is a protein with an unknown function that is similar to the yeast and human peroxisomal targeting receptor Pex5. We demonstrate that Ymr018w is a new peroxisomal-targeting receptor that targets a subset of matrix proteins to peroxisomes. We, therefore, renamed Ymr018w, Pex9, and suggest that Pex9 is a condition-specific targeting receptor that enables the dynamic rewiring of peroxisomes in response to metabolic needs. Moreover, we suggest that Pex5-like receptors might also exist in vertebrates.