Biology subjects

Chang, F.

Publications and source records attributed to Chang, F..

4 recordsLinked to original sources

bioRxiv2018

Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of Tumor Overexpressed Gene (TOG) domains. The underlying mechanism of these arrays as microtubule polymerases remains unknown. Here, we describe the biochemical and structural basis for TOG domain arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 arrays, each with distinct exchange rates. X-ray structures reveal pseudo-dimeric square-shaped assemblies in which four TOG domains position four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking confirms square assemblies form in solution, and inactivation of their interfaces destabilizes square organizations without influencing tubulin binding. Using an approach to modulate tubulin polymerization, we determined a X-ray structure showing an unfurled assembly in which TOG1 and TOG2 uniquely bind two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies, which then unfurl facilitating their concerted polymerization into protofilaments.

biochemistry↗

bioRxiv2018

Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

The XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four {beta}-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two {beta}-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that {beta}-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.

biochemistry↗

bioRxiv2018

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin - SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 - correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly bind euchromatic DNA replication origins and modify their local chromatin environment.

genetics↗

bioRxiv2017

Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

Vipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

cell biology↗