Sardana, R. | Search bioRxiv

bioRxiv2026

Cystinosin/Ers1 functions in redox homeostasis in the early secretory pathway

Cystinosis is an autosomal recessive inherited disorder caused by mutations in the CTNS gene, which encodes the highly conserved transmembrane protein cystinosin, a proton/cystine co-transporter at the lysosome membrane. However, reduction of cystine load in the lysosomes is insufficient to treat key disease symptoms, indicating that cystinosin performs additional disease-relevant functions. Here, we report that Ers1, the yeast homolog of cystinosin, localizes to and functions in the early secretory pathway. We provide evidence that Ers1 does not transport cystine. Ers1 genetically interacts with early secretory pathway recycling adaptors and redox-active Fe-S cluster-binding proteins. Notably, cystinosin-LKG, the extra-lysosomal localized splicing isoform of cystinosin, can functionally replace Ers1 in yeast. Collectively, our work uncovers a conserved role of cystinosin/Ers1 in the early secretory pathway, offering new molecular insights for understanding cystinosis pathology.

cell biology↗

bioRxiv2021

Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

cell biology↗

bioRxiv2020

Bud23 promotes the progression of the Small Subunit Processome to the pre-40S ribosome in Saccharomyces cerevisiae

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not well understood. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of 18S rRNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23{Delta}-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the suppressing mutations weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the GTPase Bms1 and the RNA helicase Dhr1. Bms1 is thought to drive conformational changes to promote rRNA cleavage, and we previously showed that Dhr1 is required for unwinding the U3 snoRNA. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and pre-rRNAs not cleaved at sites A1 and A2. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression. Author summaryRibosomes are the molecular machines that synthesize proteins and are composed of a large and a small subunit which carry out the essential functions of polypeptide synthesis and mRNA decoding, respectively. Ribosome production is tightly linked to cellular growth as cells must produce enough ribosomes to meet their protein needs. However, ribosome assembly is a metabolically expensive pathway that must be balanced with other cellular energy needs and regulated accordingly. In eukaryotes, the small subunit (SSU) Processome is a metastable intermediate that ultimately progresses towards a mature SSU through the release of biogenesis factors. The decision to progress the SSU Processome is thought to be an early checkpoint in the SSU assembly pathway, but what drives this checkpoint is unknown. Previous studies suggest that Bud23 plays an uncharacterized role during SSU Processome progression. Here, we used a genetic approach to understand its function and found that Bud23 is connected to a network of factors that stabilize the particle. Interestingly, two of these factors are enzymes that facilitate structural rearrangements needed for progression. We conclude that Bud23 promotes the release of factors surrounding its binding site to drive rearrangements during the progression of the SSU Processome.

molecular biology↗