The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase

doi:10.1126/science.abc5809

GSTDTAP > 气候变化

DOI	10.1126/science.abc5809
	The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase
	Michael J. McKenna; Sue Im Sim; Alban Ordureau; Lianjie Wei; J. Wade Harper; Sichen Shao; Eunyong Park
	2020-09-25
发表期刊	Science
出版年	2020
英文摘要	Mistargeting and misinsertion of membrane proteins causes proteostasis stress and dysfunction of intracellular organelles, which can lead to disease. McKenna et al. found that a conserved orphan P-type adenosine triphosphatase (ATPase) transporter removes misinserted transmembrane segments from the endoplasmic reticulum (ER). Functional reconstitutions and cryo–electron microscopy structures show how this ATPase selectively extracts mitochondrial proteins that are mistargeted to the ER and transmembrane segments that are inserted in the wrong orientation. This work identifies polypeptides as a new class of P-type ATPase substrates and defines a new protein quality-control mechanism at the ER. Science , this issue p. [eabc5809][1] ### INTRODUCTION Eukaryotic cells contain membrane-bound organelles with distinct identities and functionalities that depend on protein composition. Correct localization of proteins is thus critical for organelle function and cellular homeostasis. The endoplasmic reticulum (ER) and mitochondrial outer membrane are the primary destinations for newly synthesized proteins with hydrophobic transmembrane segments (TMs). Membrane protein localization requires not only high-fidelity protein targeting but also quality control mechanisms that selectively remove mislocalized proteins. At the mitochondrial outer membrane, the ATP-dependent motor protein Msp1/ATAD1 removes some mislocalized transmembrane proteins. By contrast, although protein targeting to the ER is well studied, the mechanisms that remove mistargeted transmembrane proteins from the ER membrane are incompletely understood. ### RATIONALE As a model to study membrane protein localization, we focused on tail–anchored proteins, which contain a single C-terminal TM that is necessary and largely sufficient for organelle localization. We reasoned that factors that mediate mitochondrial tail–anchored protein localization would interact directly with nascent proteins. We used an unbiased, site-specific cross-linking and mass spectrometry approach to identify such protein TMs. This approach revealed that the ER-resident orphan P-type pump P5A-ATPase (Spf1 in yeast; ATP13A1 in humans) interacted directly with a mitochondrial tail–anchored protein. Because genetic studies have linked the P5A-ATPase to mitochondrial tail–anchored protein mislocalization, we combined biochemical and structural approaches to define the function and mechanism of the P5A-ATPase. ### RESULTS P-type ATPases form a large class of active transporters that are present in all kingdoms of life and predominantly transport ions or lipids across cellular membranes. The P5A-ATPase belongs to a eukaryotic-specific subfamily of P-type ATPases with unknown substrate specificity. We reconstituted membrane protein insertion into organelles in a cell-free system and used site-specific cross-linking to reveal that the P5A-ATPase interacts directly with the TM of a mitochondrial tail–anchored protein. Human cells lacking ATP13A1 showed mislocalization of mitochondrial tail–anchored proteins to the ER and secretory pathway. In in vitro assays, newly synthesized mitochondrial tail–anchored proteins aberrantly accumulated in ER vesicles lacking P5A-ATPase activity. This accumulation was due to the impaired extraction of misinserted mitochondrial proteins from ER membranes lacking ATP13A1. Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 in different conformations at 3.3 to 3.7 Å resolutions revealed that the P5A-ATPase has an atypically large substrate-binding pocket compared with other P-type ATPases with known structures. The pocket alternately opens toward the ER lumen and cytosol while remaining accessible to the lipid bilayer through a lateral opening. Trapping putative substrates for structure determination revealed an additional membrane-spanning density at the lateral opening, which resembles an α-helical TM. Together with proteomics of wild-type and P5A-ATPase knock-out cells, our results indicate that the P5A-ATPase can dislocate moderately hydrophobic TMs with short hydrophilic lumenal domains that misinsert into the ER. ### CONCLUSION Our findings define the function of the P5A-ATPase as a dislocase of TMs at the ER membrane. This assignment establishes polypeptides as P-type ATPase transport substrates in addition to ions and lipids. Active dislocation of misinserted proteins from the ER by the P5A-ATPase also represents a previously unknown cellular safeguarding and quality control mechanism that helps maintain ER and mitochondrial homeostasis, possibly explaining the pleiotropic phenotypes linked to P5A-ATPase dysfunction. ![Figure][2] P5A-ATPase dislocates mistargeted TMs from the ER. ( A ) Diagram of a eukaryotic cell showing the nucleus (blue), ER (pale green), and mitochondria (pale purple). ( B ) Immunofluorescence images showing mislocalization of a mitochondrial tail–anchored protein containing the mitochondrial TM OMP25 (green) in P5A-ATPase knock-out cells. A mitochondrial marker (TOM20) is shown in purple. ( C ) Model for 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 representations) and the position of a substrate TM (green ribbon) bound to the outward-open form. Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces 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. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis. [1]: /lookup/doi/10.1126/science.abc5809 [2]: pending:yes
领域	气候变化 ; 资源环境
URL	查看原文
引用统计	被引频次：91[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/296496
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Michael J. McKenna,Sue Im Sim,Alban Ordureau,et al. The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase[J]. Science,2020.
APA	Michael J. McKenna.,Sue Im Sim.,Alban Ordureau.,Lianjie Wei.,J. Wade Harper.,...&Eunyong Park.(2020).The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase.Science.
MLA	Michael J. McKenna,et al."The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase".Science (2020).