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Mckenna, Sim Et Al., Science, 2020 RESEARCH ◥ zation, we combined biochemical and structural RESEARCH ARTICLE SUMMARY approaches to define the function and mecha- nism of the P5A-ATPase. STRUCTURAL BIOLOGY RESULTS: P-type ATPases form a large class of The endoplasmic reticulum P5A-ATPase is a active transporters that are present in all kingdoms of life and predominantly trans- transmembrane helix dislocase port ions or lipids across cellular membranes. The P5A-ATPase belongs to a eukaryotic- Michael J. McKenna*, Sue Im Sim*, Alban Ordureau, Lianjie Wei, J. Wade Harper, specific subfamily of P-type ATPases with un- Sichen Shao†, Eunyong Park† known substrate specificity. We reconstituted membrane protein insertion into organelles in a cell-free system and used site-specific cross- INTRODUCTION: Eukaryotic cells contain geted transmembrane proteins from the ER linking to reveal that the P5A-ATPase interacts membrane-bound organelles with distinct membrane are incompletely understood. directly with the TM of a mitochondrial tail– identities and functionalities that depend anchored protein. Human cells lacking ATP13A1 on protein composition. Correct localization RATIONALE: As a model to study membrane pro- showed mislocalization of mitochondrial tail– of proteins is thus critical for organelle func- tein localization, we focused on tail–anchored anchored proteins to the ER and secretory tion and cellular homeostasis. The endoplas- proteins, which contain a single C-terminal TM pathway. In in vitro assays, newly synthesized mic reticulum (ER) and mitochondrial outer that is necessary and largely sufficient for or- mitochondrial tail–anchored proteins aber- Downloaded from membrane are the primary destinations for ganelle localization. We reasoned that factors rantly accumulated in ER vesicles lacking newly synthesized proteins with hydrophobic that mediate mitochondrial tail–anchored pro- P5A-ATPase activity. This accumulation was transmembrane segments (TMs). Membrane tein localization would interact directly with due to the impaired extraction of misinserted protein localization requires not only high- nascent proteins. We used an unbiased, site- mitochondrial proteins from ER membranes fidelity protein targeting but also quality con- specific cross-linking and mass spectrome- lacking ATP13A1. Cryo–electron microscopy trol mechanisms that selectively remove try approach to identify such protein TMs. structures of Saccharomyces cerevisiae Spf1 mislocalized proteins. At the mitochondrial This approach revealed that the ER-resident in different conformations at 3.3 to 3.7 Å res- http://science.sciencemag.org/ outer membrane, the ATP-dependent motor orphan P-type pump P5A-ATPase (Spf1 in yeast; olutions revealed that the P5A-ATPase has an protein Msp1/ATAD1 removes some mislocal- ATP13A1 in humans) interacted directly with a atypically large substrate-binding pocket ized transmembrane proteins. By contrast, al- mitochondrial tail–anchored protein. Because compared with other P-type ATPases with though protein targeting to the ER is well genetic studies have linked the P5A-ATPase to known structures. The pocket alternately opens studied, the mechanisms that remove mistar- mitochondrial tail–anchored protein mislocali- toward the ER lumen and cytosol while remain- ing accessible to the lipid bilayer through a lateral opening. Trapping putative substrates for structure determination revealed an addi- A C tional membrane-spanning density at the lat- Cytosol Mitochondria eral opening, which resembles an a-helical TM. on January 4, 2021 Together with proteomics of wild-type and P5A-ATPase knock-out cells, our results indi- Mitochondrial TM cate that the P5A-ATPase can dislocate mode- rately hydrophobic TMs with short hydrophilic lumenal domains that misinsert into the ER. Mistargetingg CONCLUSION: Our findings define the func- tion of the P5A-ATPase as a dislocase of TMs N at the ER membrane. This assignment estab- B A lishes polypeptides as P-type ATPase transport P substrates in addition to ions and lipids. Ac- TM tive dislocation of misinserted proteins from the ER by the P5A-ATPase also represents a previously unknown cellular safeguarding and quality control mechanism that helps main- tain ER and mitochondrial homeostasis, pos- WT P5A-ATPase P5A-ATPase ATP Inward-open sibly explaining the pleiotropic phenotypes Mitochondrial TM KO Outward-open hydrolysis (E1 form) linked to P5A-ATPase dysfunction.▪ Mitochondria ER lumen (E2 form) The list of author affiliations is available in the full article online. P5A-ATPase dislocates mistargeted TMs from the ER. (A) Diagram of a eukaryotic cell showing the *These authors contributed equally to this work. nucleus (blue), ER (pale green), and mitochondria (pale purple). (B) Immunofluorescence images showing †Corresponding author. E-mail: [email protected] mislocalization of a mitochondrial tail–anchored protein containing the mitochondrial TM OMP25 (green) (S.S.); [email protected] (E.P.) Cite this article as M. J. McKenna et al., Science 369, in P5A-ATPase knock-out cells. A mitochondrial marker (TOM20) is shown in purple. (C) Model for eabc5809 (2020). DOI: 10.1126/science.abc5809 P5A-ATPase–mediated removal of mistargeted TMs from the ER membrane based on cryo–electron microscopy structures showing different conformations of the yeast P5A-ATPase (Spf1; surface READ THE FULL ARTICLE AT representations) and the position of a substrate TM (green ribbon) bound to the outward-open form. https://doi.org/10.1126/science.abc5809 McKenna et al., Science 369, 1583 (2020) 25 September 2020 1of1 RESEARCH ◥ protein containing the mitochondrial OMP25 RESEARCH ARTICLE TM during reconstituted targeting reactions (Fig.1A).Weincorporatedtheultraviolet STRUCTURAL BIOLOGY (UV) light–activated cross-linker p-benzoyl-L- phenylalanine (Bpa) into the TM [referred to The endoplasmic reticulum P5A-ATPase is a as TA(Bpa)] and purified recombinant, FLAG- tagged TA(Bpa) in complex with calmodulin transmembrane helix dislocase (CaM), a calcium-dependent TM chaperone (fig. S1A) (22, 23). We confirmed that FLAG- Michael J. McKenna1*, Sue Im Sim2*, Alban Ordureau1, Lianjie Wei1†, J. Wade Harper1, TA(Bpa) released from CaM by EGTA formed Sichen Shao1‡, Eunyong Park2,3‡ UV-dependent cross-links to the TM chaper- one SGTA (24)(Fig.1Bandfig.S1C).Whenwe Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized released FLAG-TA(Bpa) in the presence of and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine crude yeast membranes that could recon- triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane stitute TM insertion (fig. S1B), we observed segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of cross-links to various proteins. Because photo- mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of cross-linking generates covalent adducts, we Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that expected this approach to enrich for otherwise alternately faced the ER lumen and cytosol and an endogenous substrate resembling an a-helical TM. transient interactions made by substrates un- Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short dergoing targeting or QC. Indeed, tandem Downloaded from basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type mass tag mass spectrometry (TMT-MS) anal- ATPase substrates and may correct mistakes in protein targeting or topogenesis. ysis of UV-dependent membrane interactors of FLAG-TA(Bpa) identified known mitochon- drial protein receptors and QC factors (Fig. 1C, ore than one-third of the proteome in changes driven by phosphorylation and dephos- fig. S1D, and data S1) (25–27). Unexpectedly, eukaryotes consists of membrane and phorylation of a conserved aspartate (5–7). the ER-resident P5A-ATPase Spf1 was a promi- secretory proteins with diverse hydro- Among the five subfamilies categorized by nent UV-dependent interactor of FLAG-TA(Bpa) http://science.sciencemag.org/ M phobic sequences that must insert sequence similarity, the functions of P1- to (Fig. 1, C and D). correctly into the appropriate cellular P3-ATPases as cation transporters and P4- Although Spf1 has been linked to mitochon- membrane. Protein mistargeting causes or- ATPases as lipid flippases are well defined. drial TA protein mislocalization (4, 11), a direct ganelle dysfunction and cellular and organis- In addition, ATP13A2, one of four mamma- interaction has not been reported. To confirm mal stress (1, 2), underscoring the importance lian P5B-ATPases, was recently proposed to the interaction between the P5A-ATPase and TA of quality control (QC) mechanisms that re- be a lysosomal polyamine transporter (8). proteins, we translated radiolabeled TA(Bpa) move mislocalized proteins. Defined by a single Otherwise, little is known about the substrates in vitro and performed site-specific cross- C-terminal transmembrane segment (TM), tail- and biological functions of P5-ATPases. linking with crude yeast membranes or human anchored (TA) proteins pose a distinct sorting Humans and yeast each have a single P5A- ER-derived rough microsomes (RMs) contain- challenge because they rely exclusively on post- ATPase, ATP13A1 and Spf1, respectively, which ing FLAG-tagged Spf1 or ATP13A1, respective-
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