One of the hallmarks of eukaryotic cells is their organization into membrane-bound compartments, known as organelles. Some major organelles include the endoplasmic reticulum (ER), nucleus, Golgi apparatus, mitochondria, and endosomes and lysosomes. The purpose of these subcellular compartments is to provide specialized environments where specific biological reactions and processes can take place. Thus, in order to perform their essential physiological roles, organelles must maintain their own characteristic composition. While a membrane lipid bilayer can serve as a physical barrier from the cytosol, dedicated machineries must also exist to transport molecules across this membrane so that organelles can still communicate with the rest of the cell. These transmembrane transporters recognize specific substrates and directionally transport them across the membrane, while preserving the permeability barrier. Here, we describe the characterization of three different transporters using both structural biology and biochemistry: (1) the TIM23 mitochondrial protein translocase, (2) the P5A-ATPase transmembrane helix dislocase, and (3) the P5B-ATPase polyamine transporter.

Protein sorting and trafficking is essential for establishing the identity of organelles and ensuring that proteins reach their destination within the cell to perform their proper functions. A major step during this process requires the transport of proteins across or their integration into the membranes of organelles. The protein complexes responsible for catalyzing this step are known as translocases. While the process of protein translocation at the ER is well-characterized through foundational studies of the Sec61 complex, mitochondrial protein translocation is comparatively less understood. The conserved TIM23 translocase is known to import proteins into the mitochondrial matrix and inner membrane, but its mechanism has remained unclear due to a lack of structural information. Here, we have determined the cryo-electron microscopy (cryo-EM) structure of the core TIM23 complex (heterotrimeric Tim17–Tim23–Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that surprisingly, the cavity of Tim17, not Tim23, forms the protein translocation path whereas Tim23 is likely to play a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

Protein localization relies not only on high-fidelity protein targeting but also quality control mechanisms that selectively remove mislocalized proteins. Although protein targeting to the ER is well studied, the mechanisms that remove mistargeted transmembrane proteins from the ER membrane are incompletely understood. We found that the ER-resident orphan P5A-ATPase transporter (Spf1 in yeast and ATP13A1 in mammals) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored (TA) proteins. The activity of the P5A-ATPase mediated the extraction of mistargeted proteins from the ER. Cryo-EM structures of S. cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Together with proteomics studies, our results indicate that the P5A-ATPase can remove moderately hydrophobic TMs with short hydrophilic lumenal domains that misinsert into the ER. Our findings establish polypeptides as substrates of the P5A-ATPase and define the function of this transporter as a dislocase of TMs at the ER membrane. This newly assigned role of the P5A-ATPase represents a previously unknown cellular safeguarding and quality control mechanism that helps maintain ER homeostasis.

Related to the P5A-ATPase, the P5B-ATPases belong to the same subfamily of active transporters within the P-type ATPase superfamily. The P5 subfamily had historically remained the least characterized among P-type ATPases. Interest in these transporters increased when loss-of-function mutations in the human P5B-ATPase ATP13A2 were found to cause hereditary early-onset Parkinson’s disease, called Kufor Rakeb syndrome. Recently, studies discovered that the P5B-ATPases function as polyamine transporters at endo-/lysosomes. Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not well-understood. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryo-EM structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand the functions and mechanisms of P5B-ATPases.