scholarly journals A TRCky TA protein delivery service snubs the UPS

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Alexander J. McQuown ◽  
Dvir Reif ◽  
Vladimir Denic
Keyword(s):  
Protein Delivery ◽  
Proteasomal Degradation ◽  
Hek293 Cells ◽  
Membrane Insertion ◽  
Delivery Service ◽  
Er Targeting ◽  
Ta Proteins ◽  
Er Membrane

In mammals, tail-anchored (TA) proteins that are posttranslationally captured by the chaperone SGTA are triaged by the BAG6 complex into one of two fates: handoff to an ER targeting factor for membrane insertion or polyubiquitination for destruction by the proteasome. In this issue, Culver and Mariappan (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202004086) show that a fraction of newly synthesized TA proteins is polyubiquitinated in HEK293 cells independently of the BAG6 complex yet evades proteasomal degradation by undergoing deubiquitination en route to becoming stably inserted into the ER membrane.

Proteomic Analysis of the alphaIIbbeta3 Interactome Reveals Novel Chaperone and Trafficking Proteins, Including An HSP40 Chaperone, DNAJC10, That Regulates alphaIIbbeta3 Surface Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2863-2863
Author(s):  
Amanda Chen ◽  
Mahmoud Yazdani-Abyaneh ◽  
W. Beau Mitchell
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Protein Delivery ◽  
Uv Light ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Physical Interaction ◽  
Granule Formation ◽  
Angiogenic Proteins ◽  
Proplatelet Formation

Abstract Platelet alpha granules, which contain both membrane-bound and secreted proteins, are formed in the megakaryocyte and then delivered along proplatelet elaborations to the newly forming platelets. A critical but poorly understood process is the post-translational processing, sorting and delivery of proteins to alpha granules prior to their delivery to the proplatelet. Defects in the processing and trafficking of platelet proteins can result in reduced platelet formation. In addition, platelet alpha granules may be differentially packaged with pro- or anti-angiogenic proteins, suggesting that protein sorting events that occur before alpha granule formation may significantly impact platelet end-point function. Thus, research at the intersection of protein trafficking and thrombopoiesis may lead to clearer understanding of the mechanisms of proplatelet formation, the mechanisms of disease in inherited platelet disorders, and the mechanisms of platelet function in inflammation, tumor metastasis and angiogenesis. We have used the platelet integrin aIIbb3 as a model protein to investigate the mechanisms of these processes. In this study we identified proteins that interacted with aIIb in either HEK293 cells or stem cell derived megakaryocytes. The purpose of expanding the “interactome” of aIIb is to identify novel protein-protein interactions that are important for protein delivery to the megakaryocyte surface, and thus could be important for alpha granule and proplatelet formation. We used two methods of capturing interacting proteins: 1) a two-cell pull-down assay using Histidine-tagged aIIb and b3 as bait for umbilical cord blood derived megakaryocyte lysate, followed by nickel bead extraction, 2) a crosslinking assay in which photoreactive, crosslinking amino acids are incorporated into growing megakaryocytes, then crosslinked by exposure to UV light. To enrich the population of aIIb residing in the ER and Golgi, a mutant aIIb subunit containing a R858G mutation, which prevents cleavage of pro-aIIb to mature aIIb resulting in intracellular retention, was used as bait in some assays. The captured proteins from both methods were separated by SDS-PAGE and analyzed by mass spectroscopy. Two or more unique peptides were identified for 93 proteins, and 33 proteins were identified in two or more separate experiments. Of these proteins, 45 were potential protein-trafficking proteins, known to interact with aIIb, or of unknown function. Further analysis of one of these proteins, DNAJC10, suggested that it plays a role in aIIbb3 biogenesis and trafficking. DNAJC10 is an HSP40 type protein with a BiP binding domain and a second domain containing two disulfide isomerase motifs. It has been shown to be induced during ER stress, and may assist in delivering misfolded ER proteins to the proteasome for degradation. Immunoprecipitation of aIIb and b3 followed by immunoblot with anti-DNAJC10 mAb revealed protein bands corresponding to the molecular mass of DNAJC10, indicating direct or indirect physical interaction of aIIb and b3 with DNAJC10. siRNA mediated knockdown of DNAJC10 increased aIIbb3 surface expression on human megakaryocytes by 12%. Together these findings indicate that DNAJC10 interacts with aIIbb3 and may play a role in regulating aIIbb3 surface expression. This study offers new insights into the control of aIIbb3 surface expression and further studies may reveal new targets for anti- or pro-integrin therapies.

On the road to nowhere: cross-talk between post-translational protein targeting and cytosolic quality control

2016 ◽  
Vol 44 (3) ◽  
pp. 796-801 ◽  
Author(s):  
Joseph Casson ◽  
Michael McKenna ◽  
Stephen High
Keyword(s):  
Quality Control ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Decisive Role ◽  
Proteasomal Degradation ◽  
Tetratricopeptide Repeat ◽  
The Road ◽  
Precursor Proteins ◽  
On The Road ◽  
Ta Proteins

A well-defined co-translational pathway couples the synthesis and translocation of nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), thereby minimizing the possibility of the hydrophobic signals and transmembrane domains that such proteins contain from being exposed to the cytosol. Nevertheless, a proportion of these co-translational substrates may fail to reach the ER, and therefore mislocalize to the cytosol where their intrinsic hydrophobicity makes them aggregation-prone. A range of hydrophobic precursor proteins that employ alternative, post-translational, routes for ER translocation also contribute to the cytosolic pool of mislocalized proteins (MLPs). In this review, we detail how mammalian cells can efficiently deal with these MLPs by selectively targeting them for proteasomal degradation. Strikingly, this pathway for MLP degradation is regulated by cytosolic components that also facilitate the TRC40-dependent, post-translational, delivery of tail-anchored membrane proteins (TA proteins) to the ER. Among these components are small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and Bcl-2-associated athanogene 6 (BAG6), which appear to play a decisive role in enforcing quality control over hydrophobic precursor proteins that have mislocalized to the cytosol, directing them to either productive membrane insertion or selective ubiquitination and proteasomal degradation.

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

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.

scholarly journals Structural basis for membrane insertion by the human ER membrane protein complex

Science ◽  
2020 ◽  
pp. eabb5008 ◽  
Author(s):  
Tino Pleiner ◽  
Giovani Pinton Tomaleri ◽  
Kurt Januszyk ◽  
Alison J. Inglis ◽  
Masami Hazu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Structural Basis ◽  
Membrane Insertion ◽  
Transmembrane Helices ◽  
Membrane Protein Complex ◽  
Cryo Electron Microscopy ◽  
Membrane Thinning ◽  
Positively Charged ◽  
Er Membrane

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.

scholarly journals Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2

1997 ◽  
Vol 139 (3) ◽  
pp. 589-599 ◽  
Author(s):  
Sankar Addya ◽  
Hindupur K. Anandatheerthavarada ◽  
Gopa Biswas ◽  
Shripad V. Bhagwat ◽  
Jayati Mullick ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Signal Sequence ◽  
Limited Proteolysis ◽  
Membrane Insertion ◽  
Sequence Motif ◽  
Specific Marker ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Er Membrane

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Christian Kohl ◽  
Peter Tessarz ◽  
Karina von der Malsburg ◽  
Regina Zahn ◽  
Bernd Bukau ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Protein Sorting ◽  
Interaction Partner ◽  
Parallel Pathways ◽  
Chaperone Interactions ◽  
Ta Proteins ◽  
Tpr Domain ◽  
Er Membrane

Abstract TPR proteins modulate the activity of molecular chaperones. Here, we describe the S. cerevisiae TPR protein Sgt2 as interaction partner of Ssa1 and Hsp104 and as a component of the GET pathway by interacting with Get5. The GET pathway mediates the sorting of tail-anchored (TA) proteins, harboring a C-terminal trans-membrane segment, to the ER membrane. S. cerevisiae sgt2Δ cells show partial defects in TA protein sorting. Sgt2 activity in vivo relies on its N- and C-terminal domains, whereas the central TPR domain and thus chaperone interactions are dispensable. We show that TA protein sorting defects are more severe in sgt2Δ get5Δ mutants compared to single knockouts. Furthermore, overproduction of Sgt2 becomes toxic to get3Δ but not to get5Δ cells. Together, these findings indicate an additional, Get5-independent role of Sgt2 in TA protein sorting, pointing to parallel pathways of substrate delivery to Get3.

Orientation of K+ channel proteins in the peri-nuclear ER membrane of HEK293 cells

2011 ◽  
Vol 71 ◽  
pp. e313
Author(s):  
Yoshimichi Murata ◽  
Itsuro Kazama ◽  
Yoshio Maruyama
Keyword(s):  
Hek293 Cells ◽  
K Channel ◽  
Channel Proteins ◽  
Er Membrane
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