SummaryCytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

Centromeres are unique chromosomal loci that promote the assembly of kinetochores, macromolecular complexes that bind spindle microtubules during mitosis. In most organisms, centromeres lack defined genetic features. Rather, they are specified epigenetically by a centromere-specific histone H3 variant, CENP-A. The Mis18 complex, comprising the Mis18α:Mis18β subcomplex and M18BP1, is crucial for CENP-A homeostasis. It recruits the CENP-A-specific chaperone HJURP to centromeres and primes it for CENP-A loading. We report here that a specific arrangement of Yippee domains in a human Mis18α:Mis18β 4:2 hexamer binds two copies of M18BP1 through M18BP1’s 140 N-terminal residues. Phosphorylation by Cyclin-dependent kinase 1 (CDK1) at two conserved sites in this region destabilizes binding to Mis18α:Mis18β, limiting complex formation to the G1 phase of the cell cycle. Using an improved viral 2A peptide co-expression strategy, we demonstrate that CDK1 controls Mis18 complex recruitment to centromeres by regulating oligomerization of M18BP1 through the Mis18α:Mis18β scaffold.DOI: http://dx.doi.org/10.7554/eLife.23352.001

The microtubule depolymerase MCAK influences chromosomal instability (CIN), but what controls its activity remains unclear. Bendre et al. show that GTSE1, a protein found overexpressed in tumors, regulates microtubule stability and chromosome alignment during mitosis by inhibiting MCAK. High levels of GTSE1 are linked to chromosome missegregation and CIN.

Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore–microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

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c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attachment of heme to the cytochrome polypeptide. Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subunits, two of which (ResB and ResC) are the minimal functional unit. The ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC. Heterologous expression of ResBC in Escherichia coli lacking its own biogenesis machinery allowed us to investigate the substrate specificity of System II. ResBC is able to attach heme to monoheme c-type cytochromes c 550 from Paracoccus denitrificans and c 552 from Hydrogenobacter thermophilus, both normally matured by System I. The production of holocytochrome is enhanced by the addition of exogenous reductant. Single-cysteine variants of these cytochromes were not efficiently matured by System II, but System I was able to produce detectable amounts of AXXCH variants; this adds to evidence that there is no obligate requirement for a disulfide-bonded intermediate for the latter c-type cytochrome biogenesis system. In addition, System II was able to mature an AXXAH-containing variant into a b-type cytochrome, with implications for both heme supply to the periplasm and substrate recognition by System II

The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii, the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of ␤-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli, periplasmic chaperones SurA, Skp, and DegP work together with the ␤-barrel assembly machinery (Bam) to target and insert ␤-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system.

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