No description available.

No description available.

No description available.

No description available.

The nuclear envelope is a barrier comprised of outer and inner membranes that separate the cytoplasm from the nucleoplasm. The outer (ONM) and inner (INM) membranes have different physical characteristics and protein compositions. In contrast to the extensive data available on the protein quality control processes operating in the cytoplasm, endoplasmic reticulum and the nucleoplasm, the mechanisms controlling protein turnover at the INM are poorly documented. The work presented in this thesis focuses on Asi1, Asi2 and Asi3, three bona-fide integral INM proteins of the yeast Saccharomyces cerevisiae. By contrast to mammalian cells, yeast progress through the cell cycle with a closed mitosis, that is cells divide in the absence of the cyclical fragmentation/reassembly of the nuclear membrane. Consequently, examining the processes affecting the stability of the Asi proteins in yeast may provide useful paradigms for understanding the turnover of INM components in non-dividing, terminally differentiated and post-mitotic cells of metazoan origin.The results have contributed to the elucidation of the biological function of Asi1 and Asi3, which are homologous proteins with C-terminal RING domains. Asi1 and Asi3 function together as a dimeric E3 ubiquitin ligase complex that operates with ubiquitin conjugating enzymes Ubc6 and Ubc7. The Asi1/3 complex ubiquitylates transcription factors Stp1 and Stp2 when they gain inappropriate access to the nucleus in the absence of SPS-sensor activation. Intriguingly, the Asi1/3 complex also mediates the turnover of multiple membrane proteins that primarily localize to other cell membranes. This latter finding indicates that the barrier function of nuclear pore complexes is not as tight as previously thought. Consistently, asi1 null mutations are synthetic lethal when introduced into hrd1Δ ire2Δ cells with compromised ER-associated degradation (ERAD) and unfolded protein response (UPR) pathways. Together the results define Asi1/3 as components of a novel quality control pathway operating in association with the INM that acts to safeguard the identity and maintain the function of the nuclear compartment. Asi1 and Asi2 exhibit rapid turnover and their turnover is ubiquitin-dependent, exhibiting a clear requirement for Ubc7. The ubiquitylated forms of Asi1 and Asi2 are degraded by nuclear-localized proteasomes; the ubiquitylated forms exhibit enhanced stability in sts1-2 mutants. Asi1 turnover requires Cue1, the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs unimpeded in cells lacking a functional Asi1/3 complex and in cells lacking Doa10, an E3 ligase complex also known to function at the INM. Consequently, Asi1 is subject to a quality control pathway associated with INM but that is distinct from the Asi1/3 and Doa10 INM- associated degradative (INMAD) pathways. This thesis documents work that clearly demonstrates that the INM is a highly dynamic structure that possesses multiple and active quality control pathways. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

No description available.

The amino-terminal 33 amino acids of the baculovirus integral membrane protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced intranuclear microvesicles (MV) and occlusion derived virus envelopes during infection, and has been termed the sorting motif (SM). When abundantly expressed, SM-fusions are also detected in the inner nuclear membrane (INM), outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting proteins with the SM use the same trafficking pathway as cellular INM proteins to traffic to nuclear membranes. This study identifies the essential characteristics required for sorting of the SM to the INM of uninfected cells, and the MV and ODV envelopes of infected cells. These features are an 18 amino acid transmembrane sequence that lacks polar and charged amino acids (a.a.) with a cluster of charged a.a. spaced 5-11 residues from the end of the transmembrane sequence. A comparison of the a.a. sequence of these SM features with cellular INM proteins shows the features are conserved. The model of INM protein sorting and localization predicts the only known sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after photobleaching to compare the localization and mobility of lamin B receptor (LBR) fusions (which contain SM-like sequences) to a viral SM fusion when expressed in either mammalian or insect cells. The results show that immobilization is not necessarily required for accumulation of proteins in the INM. Furthermore, the results from infected cells show that an active sorting event, likely independent of immobilization, can distinguish the viral SM from cellular sequences similar to the SM. The results of this study show that sorting of proteins to the INM can be mediated by the viral SM or INM protein SM-like sequences that can function either independent of, or in addition to, immobilization. These data combined with recent reports suggest that in addition to diffusion:retention a signal mediated mechanism for sorting and localization to the INM can occur.

Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...

No description available.

No description available.