Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein–protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor–cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge–charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Distinct mutations in importin-β family nucleocytoplasmic transport receptors transportin-SR and importin-13 affect specific cargo binding

Importin-(Imp)β family nucleocytoplasmic transport receptors (NTRs) are supposed to bind to their cargoes through interaction between a confined interface on an NTR and a nuclear localization or export signal (NLS/NES) on a cargo. Although consensus NLS/NES sequence motifs have been defined for cargoes of some NTRs, many experimentally identified cargoes of those NTRs lack those motifs, and consensus NLSs/NESs have been reported for only a few NTRs. Crystal structures of NTR–cargo complexes have exemplified 3D structure-dependent binding of cargoes lacking a consensus NLS/NES to different sites on an NTR. Since only a limited number of NTR–cargo interactions have been studied, whether most cargoes lacking a consensus NLS/NES bind to the same confined interface or to various sites on an NTR is still unclear. Addressing this issue, we generated four mutants of transportin-(Trn)SR, of which many cargoes lack a consensus NLS, and eight mutants of Imp13, where no consensus NLS has been defined, and we analyzed their binding to as many as 40 cargo candidates that we previously identified by a nuclear import reaction-based method. The cargoes bind differently to the NTR mutants, suggesting that positions on an NTR contribute differently to the binding of respective cargoes.

Cargo competition for a dimerization interface restricts and stabilizes a bacterial protease adaptor

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle–dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.

A dimmer switch for endosome-to–cell surface recycling

Endosome-to–cell surface recycling is mediated by retromer and Snx27. In this issue, Mao et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010048) detail how endosomal protein sorting responds to external stimuli and reveal that phosphorylation of Snx27 regulates its cargo-binding function resulting in reduced endosome-to–cell surface recycling.

Cargo competition for a dimerization interface stabilizes a protease adaptor in Caulobacter crescentus

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy dependent proteases. In Caulobacter crescentus, cell-cycle dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo and how RcdA recognizes its targets is unknown. Here we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation, while providing capacity for binding a range of substrates.Significance StatementEnergy-dependent proteases broadly regulate bacterial physiology and development. Adaptor proteins tune the substrate specificity of proteases to only degrade selective substrates during the bacterial life cycle and during times of cellular stress. In the process of delivering cargo to their respective proteases, adaptor proteins are inherently protected from degradation until the delivery is complete. How protease adaptors can recognize a wide range of cargo while maintaining stringent specificity and how this process results in stabilization of adaptors remains unclear. Here, we show that direct competition for distinct regions of the dimer interface of the RcdA adaptor by its cargo protects RcdA from degradation by the ClpXP protease, and that this interface can be selectively perturbed in a rational manner with biochemical and physiological consequences.HighlightsCargo binding of RcdA cargo competes with dimerizationDimerization of RcdA is necessary for self-degradation by ClpXPRcdA can deliver either cargo or other RcdA subunits to ClpXPDifferent regions of the dimerization interface are needed for different cargo

TNPO3-mediated nuclear entry of the Rous sarcoma virus Gag protein is independent of the cargo-binding domain

Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLS) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS, which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functionality of importin α/β and importin-11 has been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 mediates nuclear entry of Gag and directly binds to Gag. To our surprise, this interaction did not require the cargo-binding domain of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3 including SR-domain containing splicing factors and tRNAs that re-enter the nucleus. These results suggest that RSV hijacks the host nuclear import pathway using a unique mechanism, potentially allowing other cargo to bind TNPO3 simultaneously.ImportanceRSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the cargo binding domain of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

Molecular dissection of the Erv41-Erv46 retrograde receptor reveals a conserved cysteine-rich region in Erv46 required for retrieval activity

The retrograde cargo receptor Erv41-Erv46 retrieves non–KDEL-bearing ER resident proteins to maintain ER homeostasis. This study defines a conserved cysteine-rich element in Erv46 that is required for retrieval activity and cargo binding. We propose that disulfides within the cysteine-rich region regulate the hydrophobic cargo binding site.