Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Universal Single-Residue Terminal Labels for Fluorescent Live Cell Imaging of Microproteins.

Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.

Revisiting sORFs: overcoming challenges to identify and characterize functional microproteins.

Short ORFs (sORFs), that is, occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding ORFs by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural, and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization.

Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S.

At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.