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The Endoplasmic Reticulum P5A-Atpase Is a Transmembrane Helix Dislocase Michael J

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The Endoplasmic Reticulum P5A-Atpase Is a Transmembrane Helix Dislocase Michael J 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 http://science.sciencemag.org/ mislocalized proteins. At the mitochondrial This approach revealed that the ER-resident in different conformations at 3.3 to 3.7 Å res- 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- on September 24, 2020 Cytosol Mitochondria eral opening, which resembles an a-helical TM. 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- http://science.sciencemag.org/ secretory proteins with diverse hydro- Among the five subfamilies categorized by nent UV-dependent interactor of FLAG-TA(Bpa) 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- on September 24, 2020 challenge because they rely exclusively on post- ATPase, ATP13A1 and Spf1, respectively, which ing FLAG-tagged Spf1
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