RESUMEN
Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.
Asunto(s)
Fibroblastos , Membranas Mitocondriales , Animales , Ratones , Fibroblastos/metabolismo , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.
RESUMEN
Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Canada legalized nonmedical cannabis in October 2018, but significant variations in municipal regulations exist. This study explored the variations that exist and pondered their potential public health consequences. A comparative analysis was completed on the regulations and guidelines that addressed retailers' location and public consumption in the municipalities of Alberta, Ontario, and Québec. Municipal regulations that addressed the location of retailers were more numerous and extensive in Alberta and Ontario (in the context of provincial private retail models) than in Québec (government-based model). Municipalities in Alberta added more restrictions to public consumption laws as compared to municipalities in Ontario and in Québec. These additions were made to Alberta's and Ontario's provincial-level smoking and vaping bans which used tobacco-inspired frameworks, and to Québec's ban on smoking and vaping in all public spaces. The comparative analysis showed the importance of considering municipal cannabis regulations when studying the impact of legalization, given the significant variations that exist. Policy makers should be made aware of these variations in the regulation of cannabis in order to limit health harms and further social inequalities.
Asunto(s)
Cannabis , Alberta , Canadá , Humanos , Ontario , Salud Pública , QuebecRESUMEN
The correct targeting and insertion of tail-anchored (TA) integral membrane proteins is critical for cellular homeostasis. TA proteins are defined by a hydrophobic transmembrane domain (TMD) at their C-terminus and are targeted to either the ER or mitochondria. Derived from experimental measurements of a few TA proteins, there has been little examination of the TMD features that determine localization. As a result, the localization of many TA proteins are misclassified by the simple heuristic of overall hydrophobicity. Because ER-directed TMDs favor arrangement of hydrophobic residues to one side, we sought to explore the role of geometric hydrophobic properties. By curating TA proteins with experimentally determined localizations and assessing hypotheses for recognition, we bioinformatically and experimentally verify that a hydrophobic face is the most accurate singular metric for separating ER and mitochondria-destined yeast TA proteins. A metric focusing on an 11 residue segment of the TMD performs well when classifying human TA proteins. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem. This work provides context for previous observations and opens the door for more detailed mechanistic experiments to determine the molecular factors driving this recognition.
Asunto(s)
Retículo Endoplásmico , Eucariontes , Retículo Endoplásmico/metabolismo , Eucariontes/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Transporte de ProteínasRESUMEN
The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/fisiología , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiología , Unión Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ubiquitina/metabolismoRESUMEN
STI1-domains are present in a variety of co-chaperone proteins and are required for the transfer of hydrophobic clients in various cellular processes. The domains were first identified in the yeast Sti1 protein where they were referred to as DP1 and DP2. Based on hidden Markov model searches, this domain had previously been found in other proteins including the mammalian co-chaperone SGTA, the DNA damage response protein Rad23, and the chloroplast import protein Tic40. Here, we refine the domain definition and carry out structure-based sequence alignment of STI1-domains showing conservation of five amphipathic helices. Upon examinations of these identified domains, we identify a preceding helix 0 and unifying sequence properties, determine new molecular models, and recognize that STI1-domains nearly always occur in pairs. The similarity at the sequence, structure, and molecular levels likely supports a unified functional role.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Unión al ADN/química , Proteínas de la Membrana/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Humanos , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Asian Small-Clawed Otters (Aonyx cinerea) are a small, protected but threatened species living in freshwater. They are gregarious and live in monogamous pairs for their lifetimes, communicating via scent and acoustic vocalizations. This study utilized a hidden Markov model (HMM) to classify stress versus non-stress calls from a sibling pair under professional care. Vocalizations were expertly annotated by keepers into seven contextual categories. Four of these-aggression, separation anxiety, pain, and prefeeding-were identified as stressful contexts, and three of them-feeding, training, and play-were identified as non-stressful contexts. The vocalizations were segmented, manually categorized into broad vocal type call types, and analyzed to determine signal to noise ratios. From this information, vocalizations from the most common contextual categories were used to implement HMM-based automatic classification experiments, which included individual identification, stress vs non-stress, and individual context classification. Results indicate that both individual identity and stress vs non-stress were distinguishable, with accuracies above 90%, but that individual contexts within the stress category were not easily separable.
Asunto(s)
Nutrias/fisiología , Estrés Psicológico/fisiopatología , Vocalización Animal/fisiología , Agresión/fisiología , Animales , Ansiedad de Separación/fisiopatología , Conducta Alimentaria/fisiología , Femenino , Masculino , Espectrografía del SonidoRESUMEN
A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.
Asunto(s)
Cromosomas Artificiales de Levadura , Enzimas/genética , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/genética , Transformación Genética , Xilosa/metabolismo , Café , Medios de Cultivo/química , Etanol/metabolismo , Fermentación , Expresión Génica , Saccharomyces cerevisiae/crecimiento & desarrollo , Zea maysRESUMEN
We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.