Endoplasmic Reticulum-Shaping Atlastin Proteins Facilitate KSHV Replication

Kaposi’s sarcoma-associated herpesvirus (KSHV) has two life cycle modes: the latent and lytic phases. The endoplasmic reticulum (ER) is the site for KSHV production. Furthermore, ER stress can trigger reactivation of KSHV. Little is known about the nature of the ER factors that regulate KSHV replication. Atlastin proteins (ATLs which include ATL1, ATL2, and ATL3) are large dynamin-related GTPases that control the structure and the dynamics of the ER membrane. Here, we show that ATLs can regulate KSHV lytic activation and infection. Overexpression of ATLs enhances KSHV lytic activation, whereas ATLs silence inhibits it. Intriguingly, we find that silencing of ATLs impairs the response of cells to ER stress, and ER stress can promote the lytic activation of KSHV. Our study establishes that ATLs plays a critically regulatory role in KSHV infection, thus expanding the known scope of biological processes controlled by ATLs to include KSHV infection.

A conserved viral amphipathic helix governs the replication site-specific membrane association

Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their localizations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the alphavirus-like superfamily targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.

A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation

Endoplasmic reticulum–associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

Quantitative Proteomics and Differential Protein Abundance Analysis after the 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 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.

Folding and Insertion of Transmembrane Helices at the ER

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?

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.

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a.

Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1a. While IRE1a can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1a complexes as well as the targeting mechanism that recruits XBP1 to IRE1a oligomers, remain unknown. Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1a-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1a clusters, which only assemble upon overexpression of fluorescently-tagged IRE1a during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1a assemblies that are homogenously distributed throughout the ER membrane.