RESUMEN
Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to the canonical functions of dynein in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.
Asunto(s)
Dineínas , Proteínas de Saccharomyces cerevisiae , Dineínas/genética , Dineínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mitocondrias/metabolismo , Microtúbulos/metabolismoRESUMEN
The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.
Asunto(s)
Retículo Endoplásmico , Mitocondrias , Fosfatos de Fosfatidilinositol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Dinámicas Mitocondriales , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.
Asunto(s)
Vesículas Extracelulares , Citometría de Flujo , Vesículas Extracelulares/metabolismo , Humanos , Citometría de Flujo/métodos , Ingeniería de Proteínas/métodos , Microscopía Fluorescente/métodos , Bioingeniería/métodosRESUMEN
Mitochondrial division is critical for maintenance of mitochondrial morphology and cellular homeostasis. Previous studies have suggested that the mitochondria-ER-cortex anchor (MECA), a tripartite membrane contact site between mitochondria, the ER, and the plasma membrane, is involved in mitochondrial division. However, its role is poorly understood. We developed a system to control MECA formation and depletion, which allowed us to investigate the relationship between MECA-mediated contact sites and mitochondrial division. Num1 is the protein that mediates mitochondria-ER-plasma membrane tethering at MECA sites. Using both rapamycin-inducible dimerization and auxin-inducible degradation components coupled with Num1, we developed systems to temporally control the formation and depletion of the native contact site. Additionally, we designed a regulatable Num1-independant mitochondria-PM tether. We found that mitochondria-PM tethering alone is not sufficient to rescue mitochondrial division and that a specific feature of Num1-mediated tethering is required. This study demonstrates the utility of systems that regulate contact-site formation and depletion in studying the biological functions of membrane contact sites.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Membrana Celular/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
To better understand the mechanism of resistance caused by putative interactions between beta-tubulin and benzimidazole compounds, we sought to purify nematode-specific beta-tubulins using heterologous expression after replacement of the single Saccharomyces cerevisiae beta-tubulin gene. However, we found that haploid yeast cells containing nematode-specific beta-tubulin genes were not viable, suggesting that nematode beta-tubulin cannot substitute for the loss of the yeast beta-tubulin gene.
RESUMEN
Mitochondria make physical contact with nearly every other membrane in the cell, and these contacts have a wide variety of functions that are carried out by proteins that reside at the sites of contact. Over the past decade, tremendous insight into the identity and functions of proteins localized to mitochondrial contact sites has been gained. In doing so, it has become clear that one protein or protein complex can contribute to contact site formation and function in a wide variety of ways. Thus, complex and often surprising relationships between the roles of a mitochondrial contact site and its multifunctional resident proteins continue to be unraveled.