Regulation of a Spindle Positioning Factor at Kinetochores by SUMO-Targeted Ubiquitin Ligases.

Correct function of the mitotic spindle requires balanced interplay of kinetochore and astral microtubules that mediate chromosome segregation and spindle positioning, respectively. Errors therein can cause severe defects ranging from aneuploidy to developmental disorders. Here, we describe a protein degradation pathway that functionally links astral microtubules to kinetochores via regulation of a microtubule-associated factor. We show that the yeast spindle positioning protein Kar9 localizes not only to astral but also to kinetochore microtubules, where it becomes targeted for proteasomal degradation by the SUMO-targeted ubiquitin ligases (STUbLs) Slx5-Slx8. Intriguingly, this process does not depend on preceding sumoylation of Kar9 but rather requires SUMO-dependent recruitment of STUbLs to kinetochores. Failure to degrade Kar9 leads to defects in both chromosome segregation and spindle positioning. We propose that kinetochores serve as platforms to recruit STUbLs in a SUMO-dependent manner in order to ensure correct spindle function by regulating levels of microtubule-associated proteins.

Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

Molecular mechanisms in spindle positioning: structures and new concepts.

Coordination of cell cleavage with respect to cell geometry, cell polarity and neighboring tissues is critical for tissue maintenance, malignant transformation and metastasis. The position of the mitotic spindle within the cell determines where cell cleavage occurs. Spindle positioning is often mediated through capture of astral microtubules by motor proteins at the cell cortex. Recently, the core dynein anchor complex has been structurally resolved. Junctional complexes were shown to provide additional capture sites for astral microtubules in proliferating tissues. Finally, latest studies show that signals from centrosomes control spindle positioning and propose novel concepts for generation of centrosome identity.

Ubiquitylation regulates interactions of astral microtubules with the cleavage apparatus.

BACKGROUND: Correct positioning of the mitotic spindle relative to the cleavage apparatus is crucial for successful mitosis. In budding yeast, the Adenomatous Polyposis Coli-related protein Kar9, yeast EB1, and Myo2, a type V myosin, mediate positioning of the mitotic spindle close to the septin-anchored cleavage apparatus at the bud neck. RESULTS: We find that Kar9 is ubiquitylated and degraded by the proteasome. Ubiquitylation requires the ubiquitin-conjugating enzymes Ubc1 and Ubc4 and phosphorylation of Kar9 by yeast Cdk1. Importantly, Kar9 ubiquitylation and degradation depend on an intact cleavage apparatus. Kar9 is stabilized in septin mutant cells or cells lacking the bud neck formin Bnr1, but not in the bud formin Bni1 or the actomyosin ring. Transport of Kar9 to the bud neck by Myo2 is also required for Kar9 degradation. Abrogation of Kar9 phosphorylation and ubiquitylation increases interactions of astral microtubules (aMTs) with the bud neck and causes spindle mispositioning. Photoconversion experiments showed that Kar9 association with aMTs is stable. CONCLUSIONS: We propose that ubiquitylation controls interactions of aMTs with the cleavage apparatus through localized disassembly of Kar9 complexes.

Tsg101 is recruited by a late domain of the nucleocapsid protein to support budding of Marburg virus-like particles.

The nucleoprotein NP of Marburg virus (MARV) is the major component of the viral nucleocapsid, which also consists of the viral proteins VP35, L, and VP30, as well as the viral genome. During virus assembly at the plasma membrane, the nucleocapsids are enwrapped by the major matrix protein VP40 and the viral envelope, which contains the transmembrane glycoprotein GP. Upon recombinant expression, VP40 alone is able to induce the formation and release of virus-like particles (VLPs) that closely resemble the filamentous morphology of MARV particles. Release of these VP40-induced VLPs is partially dependent on the cellular ESCRT machinery, which interacts with a late-domain motif in VP40. Coexpression with NP significantly enhances the budding of VP40-induced VLPs by an unknown mechanism. In the present study we analyzed the impact of late domains present in NP on the release of VLPs. We observed that the ESCRT I protein Tsg101 was recruited by NP into NP-induced inclusions in the perinuclear region. In the presence of VP40, NP was then recruited to VP40-positive membrane clusters and, in turn, recruited Tsg101 via a C-terminal PSAP late-domain motif in NP. This PSAP motif also mediated a dramatically enhanced incorporation of Tsg101 into VLPs, and its deletion significantly diminished the positive effect of NP on the release of VLPs. Taken together, these data indicate that NP enhances budding of VLPs by recruiting Tsg101 to the VP40-positive budding site through a PSAP late-domain motif.

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein.

The glycosylated membrane protein M of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) is the main structural component of the virion and mediates assembly and budding of viral particles. The membrane topology of SARS-CoV M and the functional significance of its N-glycosylation are not completely understood as is its interaction with the surface glycoprotein S. Using biochemical and immunofluorescence analyses we found that M consists of a short glycosylated N-terminal ectodomain, three transmembrane segments and a long, immunogenic C-terminal endodomain. Although the N-glycosylation site of M seems to be highly conserved between group 1 and 3 coronaviruses, studies using a recombinant SARS-CoV expressing a glycosylation-deficient M revealed that N-glycosylation of M neither influence the shape of the virions nor their infectivity in cell culture. Further functional analysis of truncated M proteins showed that the N-terminal 134 amino acids comprising the three transmembrane domains are sufficient to mediate accumulation of M in the Golgi complex and to enforce recruitment of the viral spike protein S to the sites of virus assembly and budding in the ERGIC.