scholarly journals Distribution of Proteins at the Inner Nuclear Membrane Is Regulated by the Asi1 E3 Ligase in Saccharomyces cerevisiae

Genetics ◽  
2019 ◽  
Vol 211 (4) ◽  
pp. 1269-1282 ◽  
Author(s):  
Christine J. Smoyer ◽  
Sarah E. Smith ◽  
Jennifer M. Gardner ◽  
Scott McCroskey ◽  
Jay R. Unruh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
E3 Ligase ◽  
Inner Nuclear Membrane

scholarly journals Asi1 regulates the distribution of proteins at the inner nuclear membrane in Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Christine J. Smoyer ◽  
Sarah E. Smith ◽  
Scott McCroskey ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
Protein Turnover ◽  
Protein Quality ◽  
E3 Ligase ◽  
Protein Composition ◽  
Degradation Pathways ◽  
Wild Type ◽  
Inner Nuclear Membrane ◽  
Protein Levels

AbstractInner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape and stability. Mechanisms that drive changes in the INM proteome are poorly understood in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation (ERAD) and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most pronounced effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggests that Asi1 not only removes mis-targeted proteins at the INM, but it also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33. Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the NE through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.

scholarly journals A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes

2021 ◽  
Author(s):  
Shary N Shelton ◽  
Sarah E Smith ◽  
Jay R Unruh ◽  
Sue L Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
De Novo ◽  
Protein Composition ◽  
Parental Cell ◽  
Permeability Barrier ◽  
Developmentally Regulated ◽  
Inner Nuclear Membrane ◽  
Expression Of Genes ◽  
Mitotic Cells

Abstract The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport; however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. We conducted a screen to determine how the INM proteome differs between mitotic cells and gametes. In addition, we used a strategy that allowed us to determine if spores synthesize their INM proteins de novo, rather than inheriting their INM proteins from the parental cell. This screen used a split-GFP complementation system, where we were able to compare the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation, and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis. Taken together, our data provide evidence for INM changes during gametogenesis and shed light on mechanisms used to shape the INM proteome of spores.

scholarly journals A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes

2021 ◽  
Author(s):  
Shary N Shelton ◽  
Sarah E. Smith ◽  
Jay R. Unruh ◽  
Sue L. Jaspersen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
De Novo ◽  
Protein Composition ◽  
Permeability Barrier ◽  
Developmentally Regulated ◽  
Inner Nuclear Membrane ◽  
Expression Of Genes ◽  
Size Preference ◽  
Mitotic Cells

The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport, however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. Using a split-GFP complementation system, we compared the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

2020 ◽  
Vol 477 (14) ◽  
pp. 2715-2720
Author(s):  
Susana Castro-Obregón
Keyword(s):  
Cellular Senescence ◽  
Nuclear Membrane ◽  
Chromatin Structure ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Chimeric Protein ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

scholarly journals The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2.

1991 ◽  
Vol 113 (1) ◽  
pp. 13-23 ◽  
Author(s):  
G T Kitten ◽  
E A Nigg
Keyword(s):  
Nuclear Membrane ◽  
Stop Codon ◽  
Mevalonic Acid ◽  
Cysteine Residue ◽  
Inner Nuclear Membrane ◽  
Nuclear Lamins ◽  
Carboxyl Methylation ◽  
Reticulocyte Lysates ◽  
Caax Motif

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.

scholarly journals Lysine 242 within Helix 10 of the Pseudorabies Virus Nuclear Egress Complex pUL31 Component Is Critical for Primary Envelopment of Nucleocapsids

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Sebastian Rönfeldt ◽  
Barbara G. Klupp ◽  
Kati Franzke ◽  
Thomas C. Mettenleiter
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Amino Acid Position ◽  
Distal Portion ◽  
Lysine Residue ◽  
Interaction Domain ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Membrane Budding

ABSTRACT Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM), as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears to be accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation, we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 homologs were tested for localization, interaction with pUL34, and complementation of PrV-ΔUL31. We identified a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle. IMPORTANCE Vesicular transport through the nuclear envelope is a focus of research but is still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently, providing in-depth molecular details. However, how cargo is incorporated remained unclear. We identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.

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