Structure of the Sec13/31 COPII cage bound to Sec23 (2023)

Replication of positive-strand RNA viruses depends on usurped cell membranes and co-opted host proteins. Based on pharmacological inhibition as well as genetic and biochemical approaches, the authors identified a critical role for the cellular Cdc48 unfoldase/separase protein in promoting replication of tomato bushy stunt virus (TBSV). We show that TBSV infection induces the expression of Cdc48benthamianaplant. Cdc48 binds to TBSV replication proteins through its N-terminal region.in vitroTBSV replicase recombination experiments revealed that Cdc48 is required for efficient replicase assembly and activity. Surprisingly,in vitroReplication experiments also showed that excess Cdc48 facilitates the disassembly of the membrane-bound complex of viral replicase and RNA template. Cdc48 is also required for the recruitment of other host proteins. Since several human viruses, including flaviviruses, use Cdc48 (also known as VCP/p97) for replication, we hypothesized that Cdc48 might be a common holoviral host factor for plant and animal RNA viruses.

Phospholipase D (PLD) plays a critical role in regulating a variety of cellular processes, including autophagy and apoptosis. Accumulation of proteins in the lumen of the endoplasmic reticulum (ER) leads to ER stress. Although ER stress is a major cause of apoptosis and autophagy, the relationship between PLD activity and ER stress remains unclear. Protein transport from the ER to the Golgi is carried out by the transport vesicle of the mantle complex II (COPII). Here, we have shown that inhibition of PLD1 activity or PLD1 knockdown inhibits COPII vesicle transport in rat normal kidney (NRC) cells. The COPII vesicle envelope protein consists of Sar1 and the Sec23/24 and Sec13/31 complexes. For COPII vesicle formation on the ER membrane, Sar1, Sec23/24, and Sec13/31 are sequentially recruited from the cytosol to the ER membrane. Using a cell-free COPII envelope protein recruitment assay, we found that inhibition of PLD1 activity inhibits the recruitment of Sec13/31 from the cytosol to the ER membrane during COPII vesicle formation. Depletion of PLD1 in NRK cells was associated with increased expression of the ER stress marker GRP78 and apoptosis. Taken together, these results suggest that PLD1 activity regulates the trafficking of COPII vesicles from the ER to the Golgi apparatus by regulating the recruitment of Sec13/31 from the cytosol to the ER membrane during COPII vesicle formation.

metalloproteinase gp63 (Leishmania donovanigp63 (Ldgp63)) is aLeishmaniaHowever, it is unclear how newly synthesized Ldgp63 leaves the endoplasmic reticulum (ER) and is secreted by this parasite. Here we cloned, expressed and characterized the GTPase LdSar1 and other COPII components such as LdSec23, LdSec24, LdSec13 and LdSec31LeishmaniaTo understand their role in ER exit from Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds LdSec23, which in turn associates with LdSec24(1-702) to form a preembryonic complex thing. Furthermore, LdSec13 specifically interacts with His₆-LdSec31(1-603) and LdSec31 bind the pre-sprouting complex through LdSec23. Interestingly, the presence of dileucine 594/595 and valine 597 residues in the C-terminal domain of Ldgp63 are essential for binding to LdSec24 (703-966) and GFP-Ldgp63^L594A/L595Aor GFP-Ldgp63^V597SMutants can't leave the emergency room. Furthermore, budding of Ldgp63-containing COPII vesicles from the ER was regulated by LdSar1:T34N in ain vitroBudding assays revealed that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we co-expressed RFP-Ldgp63 with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that overexpression of LdSar1:T34N blocked the trafficking and secretion of Ldgp63LeishmaniaFinally, we found that LdSar1:T34N-GFP overexpressing transgenic parasites had significantly impaired survival in macrophages. Taken together, these results suggest that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63-ER export and subsequent secretion.

Cargo adapters sort transmembrane protein cargo into nascent vesicles by binding directly to their cytoplasmic domains. Recent studies have revealed previously unappreciated roles for cargo adapters and the regulatory mechanisms that govern their function. Adapter protein (AP)-1 and AP-2 clathrin adapters switch between open and closed conformations to ensure they are in the right place at the right time. Exon cargo adapters play a direct role in remodeling vesicle cleavage membranes Several different cargo adapters functioning in different trafficking pathways of the Golgi apparatus are similarly regulated by divalent binding to the ADP-ribosylation factor 1 (Arf1) GTPase, possibly allowing regulation by a threshold concentration of Arf1. Taken together, these studies suggest that freight adapters can do more than just tune freight.

The vast majority of proteins that are transported to various cellular compartments and secreted by cells require coat protein complex II (COPII) for export from the endoplasmic reticulum (ER). Many of the molecular mechanisms underlying COPII assembly are well understood in detail, but it is becoming increasingly clear that this fundamental mechanism is insufficient to explain various observed aspects of protein export from the ERlive.Here, we review recent data that advance our mechanistic understanding of COPII assembly and, in particular, how genetic disorders associated with the early secretory pathway provide fundamental insights into the regulation of ER-derived vector formation. We also highlight some open questions that future work should address to better understand the physiology of COPII-mediated trafficking.

Archives of Biochemistry and Biophysics, Band 573, 2015, S. 23-31

Several human polyomaviruses, including JCV, BKV, and TSV, have been associated with disease, especially in immunocompromised patients. While the large T antigen (LT) encoded by simian polyomavirus SV40 is well-studied and has intrinsic ATPase and DNA helicase activities, the LT of human polyomaviruses is relatively uncharacterized. To assess whether these enzymatic activities required for viral DNA replication are conserved among polyomaviruses, we performed a comparative study using LT of JCV, TSV and SV40. Purified Zn-ATPase domains of three LTs were targeted for ATPase and DNA helicase activities and interactions with the cellular tumor suppressor p53. We found that all Zn ATPases were active ATPases. The Zn-ATPase domain also acts as a DNA helicase, although the measured kinetic constants differ between the three proteins. Furthermore, the Zn-ATPase domain of TSV was more resistant than SV40 and JCV when tested against four small molecule ATPase inhibitors. Our results suggest that while LTs from JCV and TSV share core ATPase and DNA helicase activities, they exhibit important functional differences that likely translate into their respective abilities to infect and replicate in the host.

Journal of Biological Chemistry, Band 288, Ausgabe 19, 2013, S. 13431-13445

The ability of chaperones to overcome protein misfolding and aggregation is critical for maintaining proper protein homeostasis in all cells. The most studied depolymerase system to date is the Clp/Hsp100 family of "ATPases associated with various cellular activities" (AAA).⁺) ATPases use mechanical forces driven by ATP hydrolysis to remodel protein aggregates. The 38 kDa subunit of the chloroplast signaling particle (cpSRP43) provides an alternative system for the disassembly of large protein aggregates, using the energy of binding with its substrate protein to drive disassembly. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analysis suggest that the action of cpSRP43 can be divided into two steps according to different molecular requirements: (i) initial recognition, in which cpSRP43 specifically binds to a recognition motif displayed on the aggregate surface; (ii) ) aggregate remodeling in which highly adaptable binding interactions of cpSRP43 with the hydrophobic transmembrane domain of substrate proteins compete with packing interactions within the aggregate. This provides a useful framework for understanding the molecular mechanisms by which chaperone-binding interactions can be used to overcome protein aggregation without external energy input from ATP.

Encyclopedia of Biochemistry III, Band 5, 2021, S. 20-26

Microautophagy and macroautophagy refer to the process in which part of the cytoplasm is secreted through the cell membrane and transported to the hydrolysis chamber where it is broken down. This process happens in two ways. During macroautophagy, double- or multi-membrane vesicles called "autophagosomes" form in the cytoplasm. Upon completion, the outer membrane of the autophagosome fuses with the lysosome/vacuole or endosome. Subsequently, the inner membrane and trapped cytoplasmic material are degraded by hydrolases. Microautophagy is the direct uptake of cytoplasm by invaginations or arm-like extensions of the lysosome/vacuolar membrane. Autophagy, one of the major mechanisms for the breakdown and recycling of macromolecules, is tightly regulated by physiological and environmental influences.

Encyclopedia of Cell Biology, Band 2, 2016, S. 588-596

The cytoskeleton of bacteria and archaea is a complex protein system whose core component is a dynamic cell motor filament. They play a role in cell division, cell shape, the breakdown of DNA and the organization of other intracellular components. The protofilament structures and activities of various actin- and tubulin-like proteins of prokaryotes are very similar to their eukaryotic counterparts. A wide range of accessory proteins regulate their activities, although they do not include motor proteins. Filamentous proteins related to eukaryotic IF proteins/lamin and dynein appear to serve similar functions.

Structure, Vol. 21, No. 7, 2013, pp. 1068–1070

Ubiquitin chains can have different signal transduction outcomes depending on where they bind. in this issuestructureCastañeda and colleagues describe a new K11-diubiquitin structure and study effector recognition of proteasome-targeted substrates.

Brain Research, 1595 Band, 2015, Issues 1-9

Fused sarcoma (FUS) is a candidate gene for neurological disorders, including motor neuron disease and Parkinson's disease, as well as a variety of cancers. Recently, overexpression of FUS in a mouse model of motor neuron disease was reported. Thus, mutations that lead to changes in gene expression contribute to the development of neurodegenerative diseases. Evolutionary conservation of the genome has been used to predict key cis-acting DNA regulators of the FUS gene promoter that control transcription. The identified putative regulators were analyzed in reporter gene assays in cells and chicken embryos. Our analysis revealed that, in addition to the regulatory domain 5' of the transcription start site, an important regulatory domain is located in intron 1 of the gene itself. This intronic domain also functions in cell linesliveIn the neural tube of the chick embryo, including the developing motor neurons. Our data suggest that expression of FUS involves multiple domain interactions, including intronic domains. A better understanding of the regulation of FUS expression could elucidate how its stimulus-induced expression is associated with neurological disorders.