The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition.
The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7D cells were sickened by the mutation of genes coding for spindle pole body components and that spo7D was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7D cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7D and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment. T HE nuclear envelope is composed of distinct outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated with a unique set of proteins, some of which mediate interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and the nucleus. The inner and outer nuclear membranes fuse in the region surrounding each nuclear pore complex.In animal cells, the nuclear envelope disassembles as cells enter mitosis and reassembles upon mitotic exit. Nuclear envelope breakdown allows the association of chromosomes with spindle microtubules, which are nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the budding yeast Saccharomyces cerevisiae, undergo closed mitosis, where the nuclear envelope remains intact throughout the entire cell cycle. Closed mitosis is possible because the yeast centrosome-equivalent, the spindle pole body (SPB), is embedded in the nuclear envelope, allowing the SPB to nucleate both cytoplasmic and nuclear microtubules. SPB duplication requires a mechanism for inserting the new SPB into the nuclear envelope (reviewed in Jaspersen and Winey 2004). The new SPB begins to form in late G 1 /early S phase as satellite material deposited on the cytoplasmic face of an electron-dense region of the nuclear envelope, called the half-bridge. The satellite material matures into a dupl...
Significance Polo kinase regulates many processes during cell division and is upregulated in many cancers. During Drosophila female meiosis, the protein Matrimony inhibits Polo kinase using a noncanonical mechanism of Polo binding. Complete loss of Matrimony leads to meiotic catastrophe, and partial loss leads to chromosome missegregation. Proper Matrimony–Polo binding is required to prevent these defects, indicating that preventing Polo from phosphorylating targets is necessary for proper completion of meiosis. This finding is in contrast to mitosis where phosphorylation by Polo is usually required for cell division. This work provides important insight into developing anticancer therapeutic agents targeting Polo kinase.
In virtually all eukaryotic cells, protein bridges formed by the conserved inner nuclear membrane SUN (for Sad1-UNC-84) domain-containing proteins and their outer nuclear membrane binding partners span the nuclear envelope (NE) to connect the nucleoplasm and cytoplasm. These linkages are important for chromosome movements within the nucleus during meiotic prophase and are essential for nuclear migration and centrosome attachment to the NE. In Saccharomyces cerevisiae, MPS3 encodes the sole SUN protein. Deletion of MPS3 or the conserved SUN domain is lethal in three different genetic backgrounds. Mutations in the SUN domain result in defects in duplication of the spindle pole body, the yeast centrosome-equivalent organelle. A genome-wide screen for mutants that exhibited synthetic fitness defects in combination with mps3 SUN domain mutants yielded a large number of hits in components of the spindle apparatus and the spindle checkpoint. Mutants in lipid metabolic processes and membrane organization also exacerbated the growth defects of mps3 SUN domain mutants, pointing to a role for Mps3 in nuclear membrane organization. Deletion of SLP1 or YER140W/EMP65 (for ER membrane protein of 65 kDa) aggravated growth of mps3 SUN domain mutants. Slp1 and Emp65 form an ER-membrane associated protein complex that is not required directly for spindle pole body duplication or spindle assembly. Rather, Slp1 is involved in Mps3 localization to the NE.
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