N-terminal signals in the SNX-BAR paralogs Vps5 and Vin1 guide endosomal coat complex formation.

Endosomal coats incorporate membrane-binding subunits such as sorting nexin (SNX) proteins. The Saccharomyces cerevisiae SNX-BAR paralogs Vin1 and Vps5 are respective subunits of the endosomal VINE and retromer complexes whose dimerizing BAR domains are required for complex assembly and membrane association. However, a degree of promiscuity is predicted for yeast BAR-BAR pairings, and recent work has implicated the unstructured N-terminal domains of Vin1 and Vps5 in coat formation. Here, we map N-terminal signals in both SNX-BAR paralogs that contribute to the assembly and function of two distinct endosomal coats in vivo. Whereas Vin1 leverages a polybasic region and adjacent hydrophobic motif to bind Vrl1 and form VINE, the N-terminus of Vps5 interacts with the retromer subunit Vps29 at two sites, including a conserved hydrophobic pocket in Vps29 that engages other accessory proteins in humans. We also examined the sole isoform of Vps5 from the milk yeast Kluyveromyces lactis and found that ancestral yeasts may have used a nested N-terminal signal to form both VINE and retromer. Our results suggest that the specific assembly of Vps5-family SNX-BAR coats depends on inputs from unique N-terminal sequence features in addition to BAR domain coupling, expanding our understanding of endosomal coat biology.

You can go your own way: SNX-BAR coat complexes direct traffic at late endosomes.

The endolysosomal network consists of highly dynamic membrane-bound compartments that control subcellular degradative and recycling processes. A conserved family of endosomal coat complexes known as SNX-BARs drive the formation of tubular membrane transport carriers for cargo retrieval. Whereas SNX1-related SNX-BARs were previously thought to rely on their association with the retromer complex to recognize cargo, recent work shows this class of SNX-BARs can directly bind and deliver cargo. In this review, we examine the retromer-independent roles of SNX-BAR proteins in yeast and metazoans and explore their functional overlap with endosomal sorting complexes and accessory factors. We also discuss new work that highlights the role of the disordered N-terminal regions of SNX-BARs in complex assembly and function.

The VINE complex is an endosomal VPS9-domain GEF and SNX-BAR coat.

Membrane trafficking pathways perform important roles in establishing and maintaining the endosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX-BAR-containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9-domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due to an inactivating mutation found in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX-BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX-BAR protein Vps5. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming a complex that we have named VINE. The VINE complex occupies endosomal tubules and redistributes a conserved mannose 6-phosphate receptor-like protein from endosomes. We also find that membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

All healthy cells have a highly organized interior: different compartments with specialized roles are in different places, and in order to do their jobs properly, proteins need to be in the right place. Endosomes are membrane-bound compartments that act as transport hubs where proteins are sorted into small vesicles and delivered to other parts of the cell. Two groups of proteins regulate this transport: the first group, known as VPS9 GEFs, switches on the enzymes that recruit the second group of proteins, called the sorting nexins. This second group is responsible for forming the transport vesicles via which proteins are distributed all over the cell. Defects in protein sorting can lead to various diseases, including neurodegenerative conditions such as Parkinson's disease and juvenile amyotrophic lateral sclerosis. Scientists often use budding yeast cells to study protein sorting, because these cells are similar to human cells, but easier to grow in large numbers and examine in the laboratory. Previous work showed that a yeast protein called Vrl1 is equivalent to a VPS9 GEF from humans called VARP. However, Vrl1 only exists in wild forms of budding yeast, and not in laboratory strains of the organism. Therefore, researchers had not studied Vrl1 in detail, and its roles remained unclear. To learn more about Vrl1, Shortill et al. started by re-introducing the protein into laboratory strains of budding yeast and observing what happened to protein sorting in these cells. Like VARP, Vrl1 was found in the endosomes of budding yeast. However, biochemical experiments revealed that, while human VARP binds to a protein called retromer, Vrl1 does not bind to the equivalent protein in yeast. Instead, Vrl1 itself has features of both the VPS9 GEFs and the sorting nexins. Shortill et al. also found that Vrl1 interacted with a different protein in the sorting nexin family called Vin1. In the absence of Vrl1, Vin1 was found floating around the cell, but once Vrl1 was re-introduced into the budding yeast, Vin1 relocated to the endosomes. Vrl1 uses its VPS9 GEF part to move itself to the endosome membrane, and Vin1 controls this movement, highlighting the interdependence between the two proteins. Once they are at the endosome together, Vrl1 and Vin1 help redistribute proteins to other parts of the cell. This study suggests that, like VARP, Vrl1 cooperates with sorting nexins to transport proteins. Since many previous experiments about protein sorting were carried out in yeast cells lacking Vrl1, it is possible that this process was overlooked despite its potential importance. These new findings could also help other researchers investigating how endosomes and protein sorting work, or do not work, in the context of neurodegenerative diseases.

Temperature-sensitive recombinant subtilisin protease variants that efficiently degrade molecular biology enzymes.

We used error-prone PCR to generate mutations in a subtilisin protease-encoding gene, and screened for recombinants that expressed temperature-sensitive (TS) variants. From the dozens of mutations that we detected in the recombinant genes we found that those mutations that affected aspartate-75 had the most profound effect on temperature stability. We thus focused our analysis on two variants of subtilisin C, the more heat-sensitive variant 24 (V24), with amino acid changes D75G, L234M and Q274P; and variant 25 (V25), with a single amino acid change, D75A. For V24 a two log-fold reduction in activity occurs in under 10 min at 50°C. For V25, a two log-fold reduction occurs at 60°C, a temperature that reduces the activity of the wild type enzyme by about 30%. The V24 variant fully inactivates enzymes commonly used in molecular biology research and in molecular diagnostics, and is stabilized against autolysis with propylene glycol concentrations of 10% or greater. The subtilisin variants are produced by a strain of Bacillus subtilis that lacks expression of its native secreted proteases, and the variants can be isolated from the supernatants using nickel affinity chromatography.