Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of about 10,000 different soluble and membrane proteins in humans. It involves co- or post-translational targeting of precursor polypeptides to the ER and their subsequent membrane insertion or translocation. So far, three pathways for ER targeting of precursor polypeptides plus four pathways for ER targeting of mRNAs were described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in targeting and, putatively, inserting monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose if this pathway may play a more general role in ER protein targeting, i.e. represents a fourth pathway for ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach, which involves label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells and differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3-clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices and belonging to the secretory pathway were also negatively affected by PEX3-deficiency, which may suggest compromised collagen biogenesis as a hitherto unknown contributor to organ failures in the respective Zellweger patients.

Download Full-text

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

International Journal of Molecular Sciences ◽

10.3390/ijms222313028 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13028

Author(s):

Richard Zimmermann ◽

Sven Lang ◽

Monika Lerner ◽

Friedrich Förster ◽

Duy Nguyen ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Import ◽

Protein Targeting ◽

Human Cells ◽

Protein Abundance ◽

General Role ◽

Differential Protein ◽

Er Targeting ◽

Dependent Pathway ◽

Er Membrane

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

Download Full-text

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

Molecules ◽

10.3390/molecules26123591 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3591

Author(s):

Pratiti Bhadra ◽

Stefan Schorr ◽

Monika Lerner ◽

Duy Nguyen ◽

Johanna Dudek ◽

...

Keyword(s):

Quantitative Proteomics ◽

Secretory Pathway ◽

Human Cells ◽

Ribosome Profiling ◽

Protein Abundance ◽

Label Free ◽

Differential Protein ◽

Cellular Assays ◽

Pros And Cons ◽

Er Membrane

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

Download Full-text

The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

Molecular Biology of the Cell ◽

10.1091/mbc.e13-09-0526 ◽

2014 ◽

Vol 25 (4) ◽

pp. 495-507 ◽

Cited By ~ 37

Author(s):

Maren Klinger ◽

Wenbo Wang ◽

Stefanie Kuhns ◽

Felix Bärenz ◽

Stefanie Dräger-Meurer ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Basal Body ◽

Primary Cilia ◽

Protein Targeting ◽

Somatostatin Receptor ◽

Human Cells ◽

Interacting Protein ◽

Ciliary Membrane ◽

Intracellular Signals

In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow and which assembles a gate to maintain the specific ciliary proteome. Here we characterize the function of a novel centriolar satellite protein, synovial sarcoma X breakpoint–interacting protein 2 (SSX2IP), in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using small interfering RNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290 to both satellites and the basal body. Cep290 takes a central role in gating proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, somatostatin receptor 3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome, and Rab8.

Download Full-text

Mitochondrial carrier protein overloading and misfolding induce aggresomes and proteostatic adaptations in the cytosol

Molecular Biology of the Cell ◽

10.1091/mbc.e19-01-0046 ◽

2019 ◽

Vol 30 (11) ◽

pp. 1272-1284 ◽

Cited By ~ 8

Author(s):

Yaxin Liu ◽

Xiaowen Wang ◽

Liam P. Coyne ◽

Yuan Yang ◽

Yue Qi ◽

...

Keyword(s):

Stress Responses ◽

Protein Import ◽

Protein Targeting ◽

Human Cells ◽

Mitochondrial Proteins ◽

Protein Accumulation ◽

Carrier Protein ◽

Mitochondrial Carrier ◽

Fold Reduction ◽

Mitochondrial Carrier Protein

Previous studies in yeast showed that mitochondrial stressors not directly targeting the protein import machinery can cause mitochondrial precursor overaccumulation stress (mPOS) in the cytosol independent of bioenergetics. Here, we demonstrate mPOS and stress responses in human cells. We show that overloading of mitochondrial membrane carrier, but not matrix proteins, is sufficient to induce cytosolic aggresomes and apoptosis. The aggresomes appear to triage unimported mitochondrial proteins. Interestingly, expression of highly unstable mutant variants of the mitochondrial carrier protein, Ant1, also induces aggresomes despite a greater than 20-fold reduction in protein level compared to wild type. Thus, overloading of the protein import machinery, rather than protein accumulation, is critical for aggresome induction. The data suggest that the import of mitochondrial proteins is saturable and that the cytosol is limited in degrading unimported mitochondrial proteins. In addition, we found that EGR1, eEF1a, and ubiquitin C are up-regulated by Ant1 overloading. These proteins are known to promote autophagy, protein targeting to aggresomes, and the processing of protein aggregates, respectively. Finally, we found that overexpression of the misfolded variants of Ant1 induces additional cytosolic responses including proteasomal activation. In summary, our work captured a profound effect of unimported mitochondrial proteins on cytosolic proteostasis and revealed multiple anti-mPOS mechanisms in human cells.

Download Full-text

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Download Full-text

Faculty Opinions recommendation of Quality control of ER membrane proteins by the rnf185/membralin ubiquitin ligase complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738410223.793577269 ◽

2020 ◽

Author(s):

Tom Rapoport ◽

Navdar Sever

Keyword(s):

Quality Control ◽

Membrane Proteins ◽

Ubiquitin Ligase ◽

Ubiquitin Ligase Complex ◽

Er Membrane

Download Full-text

Insertion of plastidic β-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80

The Plant Cell ◽

10.1093/plcell/koab052 ◽

2021 ◽

Author(s):

Lucia E Gross ◽

Anna Klinger ◽

Nicole Spies ◽

Theresa Ernst ◽

Nadine Flinner ◽

...

Keyword(s):

Membrane Proteins ◽

Protein Import ◽

Membrane Insertion ◽

Intermembrane Space ◽

Envelope Membrane ◽

Outer Envelope ◽

Pea Proteins ◽

Outer Envelope Membrane ◽

Correct Topology ◽

Shed Light

Abstract The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for translocation of pea proteins across the outer envelope membrane is present within the six N-terminal β-strands. This process requires the action of TOC (translocon of the outer chloroplast membrane). After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.

Download Full-text

Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

The Journal of Cell Biology ◽

10.1083/jcb.200412143 ◽

2005 ◽

Vol 169 (6) ◽

pp. 897-908 ◽

Cited By ~ 108

Author(s):

Cosima Luedeke ◽

Stéphanie Buvelot Frei ◽

Ivo Sbalzarini ◽

Heinz Schwarz ◽

Anne Spang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Proteins ◽

Diffusion Barriers ◽

Yeast Cells ◽

Dependent Manner ◽

Protein Diffusion ◽

Ultrastructural Studies ◽

Bud Neck ◽

Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

Download Full-text

LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

Cells ◽

10.3390/cells9102282 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2282

Author(s):

Björn Koos ◽

Eva Lotta Moderegger ◽

Katharina Rump ◽

Hartmuth Nowak ◽

Katrin Willemsen ◽

...

Keyword(s):

Immune Response ◽

Mitochondrial Dna ◽

Dna Methyltransferase ◽

Protein Import ◽

Mononuclear Cells ◽

Mitochondrial Protein ◽

Mitochondrial Fraction ◽

Protein Abundance ◽

Peripheral Blood Mononuclear ◽

Cellular Expression

Mitochondrial DNA (mtDNA) plays a vital role as a damage-associated molecular pattern in sepsis being able to shape the immune response. Since pathogen recognition receptors of innate immune cells are activated by demethylated DNA only, we set out to investigate the amount of DNA methyltransferase 1 (DNMT1) in mitochondria and the extent of mtDNA methylation in a human endotoxin model. Peripheral blood mononuclear cells of 20 healthy individuals were isolated from whole blood and stimulated with lipopolysaccharide (LPS) for 48 h. Subsequently, DNMT1 protein abundance was assessed in whole cells and a mitochondrial fraction. At the same time, methylation levels of mtDNA were quantified, and cytokine expression in the supernatant was measured. Despite increased cellular expression of DNMT1 after LPS stimulation, the degree of mtDNA methylation slightly decreased. Strikingly the mitochondrial protein abundance of DNMT1 was reduced by 50% in line with the lower degree of mtDNA methylation. Although only modest alterations were seen in the degree of mtDNA methylation, these strongly correlated with IL-6 and IL-10 expression. Our data may hint at a protein import problem for DNMT1 into the mitochondria under LPS stimulation and suggest a role of demethylated mtDNA in the regulation of the inflammatory immune response.

Download Full-text