Mitochondria perform central functions in cellular bioenergetics, metabolism, and signaling, and their dysfunction has been linked to numerous diseases. The available studies cover only part of the mitochondrial proteome, and a separation of core mitochondrial proteins from associated fractions has not been achieved. We developed an integrative experimental approach to define the proteome of yeast mitochondria. We classified > 3,300 proteins of mitochondria and mitochondria-associated fractions and defined 901 high-confidence mitochondrial proteins, expanding the set of mitochondrial proteins by 82. Our analysis includes protein abundance under fermentable and nonfermentable growth, submitochondrial localization, single-protein experiments, and subcellular classification of mitochondria-associated fractions. We identified mitochondrial interactors of respiratory chain supercomplexes, ATP synthase, AAA proteases, the mitochondrial contact site and cristae organizing system (MICOS), and the coenzyme Q biosynthesis cluster, as well as mitochondrial proteins with dual cellular localization. The integrative proteome provides a high-confidence source for the characterization of physiological and pathophysiological functions of mitochondria and their integration into the cellular environment.

Lipid droplets (LDs) store lipids and hence serve as energy reservoir and as a source for building-blocks for the organelle membrane systems. LD biology therefore depends on tight communication with other organelles. The unique architecture of LDs, consisting of a neutral lipid core shielded by a phospholipid-monolayer, is however an obstacle to bulk-exchange of bilayer-bounded vesicles with other organelles. In recent years, it is emerging that contact sites, places where two organelles are positioned in close proximity allowing vesicle-independent communication, are an important way to integrate LDs into the organellar landscape. However, few LD contact sites have been studied in depth and our understanding of their structure, extent and function is only starting to emerge. Here, we highlight recent findings on the functions of LD contact sites and on the proteins involved in their formation and hypothesize about the unique characteristics of the contact sites formed by these intriguing organelles. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.