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Signal Peptide Peptidase‐Like 2C Impairs Vesicular Transport And

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Signal Peptide Peptidase‐Like 2C Impairs Vesicular Transport And Article Signal peptide peptidase-like 2c impairs vesicular transport and cleaves SNARE proteins Alkmini A Papadopoulou1, Stephan A Müller2, Torben Mentrup3, Merav D Shmueli2,4,5, Johannes Niemeyer3, Martina Haug-Kröper1, Julia von Blume6, Artur Mayerhofer7, Regina Feederle2,8,9 , Bernd Schröder3,10 , Stefan F Lichtenthaler2,5,9 & Regina Fluhrer1,2,* Abstract Introduction Members of the GxGD-type intramembrane aspartyl proteases The high degree of compartmentalization in eukaryotic cells creates have emerged as key players not only in fundamental cellular a need for specific and precise protein trafficking. To meet this need, processes such as B-cell development or protein glycosylation, but cells have developed a complex system of vesicle transport that also in development of pathologies, such as Alzheimer’s disease or ensures safe sorting of cargo proteins, in particular between the dif- hepatitis virus infections. However, one member of this protease ferent compartments of the secretory pathway [1–3]. Vesicles origi- family, signal peptide peptidase-like 2c (SPPL2c), remains orphan nate from a donor membrane, translocate in a targeted manner, and and its capability of proteolysis as well as its physiological function get specifically tethered to the target membrane, before fusing with is still enigmatic. Here, we demonstrate that SPPL2c is catalytically it. Soluble N-ethylmaleimide-sensitive factor attachment protein active and identify a variety of SPPL2c candidate substrates using receptor (SNARE) proteins are known since three decades to medi- proteomics. The majority of the SPPL2c candidate substrates clus- ate specific membrane fusion [4,5]. So far, 38 SNARE proteins have ter to the biological process of vesicular trafficking. Analysis of been identified in mammalian cells and in their vast majority they selected SNARE proteins reveals proteolytic processing by SPPL2c are tail-anchored type II transmembrane proteins (type IV). Their that impairs vesicular transport and causes retention of cargo N-terminal domains face the cytosol and comprise a 60–70 amino proteins in the endoplasmic reticulum. As a consequence, the acid-long conserved “SNARE motif”, which facilitates coiled-coil integrity of subcellular compartments, in particular the Golgi, is formation [3,6,7]. For two membranes to fuse, a tetrameric SNARE disturbed. Together with a strikingly high physiological SPPL2c motif complex needs to form with proteins from the opposing expression in testis, our data suggest involvement of SPPL2cin membranes. In most cases, one vesicle-membrane SNARE acrosome formation during spermatogenesis. (v-SNARE) binds three target-membrane SNAREs (t-SNAREs) form- ing a trans-complex, which becomes a cis-complex as soon as the Keywords glycosyltransferases; intramembrane proteases; SPP/SPPL-family; membranes fuse and all proteins locate to one membrane [6]. SNARE; spermatogenesis Classification of v- and t-SNAREs is not always consistent with the Subject Categories Membrane & Intracellular Transport; Post-translational actual localization of the proteins, so a more robust way to catego- Modifications, Proteolysis & Proteomics rize them is based on the conserved glutamine (Q) or arginine (R) DOI 10.15252/embr.201846451 | Received 18 May 2018 | Revised 7 December residue within their SNARE motif. Each SNARE complex is formed 2018 | Accepted 21 December 2018 | Published online 7 February 2019 by one R-SNARE and three Q-SNAREs (Qa-, Qb-, and Qc-SNAREs). EMBO Reports (2019) 20:e46451 In most cases, the v-SNARE corresponds to the R-SNARE and the t-SNAREs to the Q-SNAREs. The N-ethylmaleimide-sensitive factor See also: J Niemeyer et al (March 2019) and HS Young & MJ Lemieux (NSF) and the NSF attachment protein (SNAP) are involved in the (March 2019) dissociation of the cis-SNARE complex. Subsequently, the incom- ing SNARE proteins are returned to their donor compartment [5]. 1 Institute for Metabolic Biochemistry, Biomedical Center (BMC), Ludwig-Maximilians University Munich, Munich, Germany 2 DZNE – German Center for Neurodegenerative Diseases, Munich, Germany 3 Biochemical Institute, Christian Albrechts University of Kiel, Kiel, Germany 4 Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel 5 Neuroproteomics, School of Medicine, Klinikum Rechts der Isar, and Institute for Advanced Study, Technical University Munich, Munich, Germany 6 Max Planck Institute of Biochemistry, Martinsried, Germany 7 Cell Biology, Anatomy III, Biomedical Center (BMC), Ludwig-Maximilians University Munich, Munich, Germany 8 Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany 9 Munich Center for Systems Neurology (SyNergy), Munich, Germany 10 Institute for Physiological Chemistry, Technische Universität Dresden, Dresden, Germany *Corresponding author. Tel: +49 89 4400 46505; E-mail: [email protected] [Correction added on 6 May 2019, after first online publication: the article title has been corrected.] ª 2019 The Authors EMBO reports 20:e46451 | 2019 1 of 21 EMBO reports SPPL2c impairs vesicular transport Alkmini A Papadopoulou et al Vesicle-associated membrane proteins (VAMPs), syntaxins, and in the protein composition of cellular compartments and in the the 25-kD synaptosomal-associated protein (SNAP25) are the main glycan structure of cellular proteins. Such changes naturally occur components of the SNARE complex. SNAP-25 represents a during spermatogenesis, a process characterized by pronounced subgroup of SNAREs, mostly Qb- and Qc-SNAREs, that are the compartmental reorganization and specific changes in glycan struc- only ones lacking a transmembrane domain and comprising two tures that result in the formation of the acrosome and the glycocalyx SNARE motifs. Syntaxins predominantly act as Qa-SNAREs, and in the mature spermatozoon [24]. Our data indicate that a strikingly VAMPs as R-SNAREs [5]. Different combinations of SNARE high expression of SPPL2c in spermatids supports these processes proteins are responsible for different intracellular trafficking during spermatogenesis. routes. In mammals, the family of GxGD-type intramembrane aspartyl proteases comprises presenilin 1 and presenilin 2, which form Results the catalytically active subunit of the Alzheimer disease-associated c-secretase complex, as well as signal peptide peptidase (SPP) and SPPL2c is catalytically active and cleaves SNARE proteins its four homologous, the SPP-like (SPPL) proteases, SPPL2a, SPPL2b, SPPL2c, and SPPL3 [8,9]. Other vertebrates, such as zebra- To identify potential candidate substrates of SPPL2c, we subjected fish, only express one representative of the SPPL2 subfamily membrane preparations from T-RexTM-293 (HEK293) cells stably suggesting that in particular the individual members of the SPPL2 expressing catalytically active SPPL2c under a doxycycline-inducible subfamily have evolved diverse functions [10]. Although SPPL2c promoter and from control cells without SPPL2c expression to exhibits a FD and a GFGD motif, which reflect the catalytic signature protein label-free quantification (LFQ) mass spectrometry. Under motifs of GxGD-type intramembrane aspartyl proteases [11], the these conditions, 4,841 proteins were identified by at least two proof of SPPL2c being a catalytically active protease is still missing, peptides, and 3,726 proteins were relatively quantified on the basis not least because to date no SPPL2c substrates have been identified. of at least three replicates of SPPL2c-expressing cells and control Due to its highly polymorphic and intron-less structure, the SPPL2C cells, and were subjected to statistical analysis. Statistical data anal- gene is even discussed to be a pseudogene [12]. ysis revealed 451 out of 917 proteins with a P-value < 0.05 that were All members of the SPP/SPPL family are multi-pass transmem- significantly altered in SPPL2c-expressing cells after false discovery brane proteins that span the membrane with nine transmembrane rate (FDR) correction for multiple hypotheses (Dataset EV1). The (TM) domains [13]. In contrast to presenilins, which only process volcano plot summarizes changes in protein abundance from cells type I (Nout)-oriented TM segments, one mutuality of all SPP/SPPL- ectopically expressing SPPL2c compared to control cells (Fig 1A). family members is their selectivity for type II (Nin) TM segments As expected, the strongest change was observed for SPPL2c, which [8]. Recently, it was shown that SPP is capable of also processing was highly enriched in SPPL2c-expressing HEK293 cells (Fig 1A). the type II-oriented tail-anchored (type IV) proteins cytochrome B5A Since SPPL2c is not expressed endogenously in HEK293 cells, the (CYB5A), ribosome-associated membrane protein 4 (RAMP4), and very high fold change determined for SPPL2c may be explained by heme oxygenase-1 (HO-1) [14,15]. Generally, GxGD-type aspartyl little carryover of SPPL2c peptides from samples of overexpressing proteases favor substrates with a short luminal domain [16–18]. cells into control samples. Because of the known substrate selectiv- Only SPPL3, which was recently shown to act as a sheddase on ity of SPP/SPPL-family [8] proteases, we only considered type II glycan-modifying enzymes in cells and in mice, constitutes an transmembrane proteins and type IV tail-anchored proteins that exception [19,20]. By proteolytic release of the active site-containing were decreased upon exogenous expression of SPPL2c as potential
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