Spatial control of avidity regulates initiation and progression of selective autophagy

Autophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

An Extended Coatomer Binding Motif in the SARS-CoV-2 Spike Protein

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify a novel extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Endomembrane systems are reorganized by ORF3a and Membrane (M) of SARS-CoV-2

The endomembrane reticulum (ER) is largely reorganized by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 ORF3a and membrane (M) protein expression affects ER-derived structures including cubic membrane and double membrane vesicles in coronavirus-infected cells; however, the molecular mechanisms underlying ER remodeling remain unclear. We introduced a 'plug and playable' proximity labeling tool (TurboID-GBP) for interactome mapping of GFP-tagged SARS-CoV-2 ORF3a and M proteins. Through mass spectrometric identification of the biotinylated lysine residue (K+226 Da) on the viral proteins using Spot-TurboID workflow, 117 and 191 proteins were robustly determined as ORF3a and M interactomes, respectively, and many, including RNF5 (E3 ubiquitin ligase), overlap with the mitochondrial-associated membrane (MAM) proteome. RNF5 expression was correlated to ORF3a ubiquitination. MAM formation and secreted proteome profiles were largely affected by ORF3a expression. Thus, SARS-CoV-2 may utilize MAM as a viral assembly site, suggesting novel anti-viral treatment strategies for blocking viral replication in host cells.

On the issue of rational use of production areas in the enterprise

This paper considers the problem of uneven loading of the enterprise's production areas, when in the first half of the year they were significantly underutilized, and in the second — considerably overloaded. The result of solving this problem is to create a flexible approach to the planning of production areas, for which a mathematical model of the assembly site has been developed. This model is a linear programming problem. Four options were considered. The cost-effective option is to fulfill all orders in one shift and gradually rent the premises throughout the year. It can be noted that in general, it is more profitable to make absolutely all orders than not to make them — this is due to the high cost of equipment, so it is not possible to sacrifice it. The only exception when it is more profitable not to make part of the orders is if the rental of production areas occurs only at the beginning of the year. In the conditions that are now in the enterprise (one cannot rent areas per year, work in one shift), it is most profitable not to make part of the orders, and this option will be the second in total costs. As a result, a mathematical model of the enterprise was obtained, which can be used on tape transport system to assess the capabilities of the enterprise. This model can be used to obtain forecast values for costs. Further, this model can be extended to all other types of products.

Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the function of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned, meshwork matrix formed matrix assembly sites around the cell periphery but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independently of cell elongation. We conclude the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites which form during the initial stages of cell-FN interactions.

Vacuolar protein Tag1 and Atg1–Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae

ABSTRACTUnder starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10–12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.