Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins.

The vertebrate kinetochore complex assembles at the centromere on alpha-satellite DNA. In humans, alpha-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to alpha-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles.

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Localization and properties of kinases in clathrin-coated vesicles from zucchini hypocotyls.

Five major polypeptides of 70, 50, 47, 19 and 17 kDa and four minor polypeptides (100, 65, 45 and 39 kDa) become phosphorylated when clathrin-coated vesicles (CCV) from zucchini hypocotyls are incubated in [gamma 32P]Mg-ATP. After dissociation with 0.5 M Tris/HCl the CCV coat polypeptides were subjected to gel filtration in order to separate clathrin triskelions from beta-adaptin-containing fractions. Only the latter bore kinase activities, with phosphorylated polypeptides of 39 kDa in addition to the 50, 19-kDa and 17-kDa polypeptides just mentioned. Heparin, an inhibitor of casein kinase II, permitted the phosphorylation of only the 19-kDa and 17-kDa polypeptides. Staurosporine, an inhibitor of protein kinase c-like activities, prevented the phosporylation of the 70-kDa polypeptide. When recombined with the triskelions the beta-adaptin fractions achieved the phosphorylation of the 45-kDa and 70-kDa polypeptides. Because of its heat stability and calcium-binding properties we interpret the 45-kDa polypeptide as being a clathrin light chain. Antibodies raised against the 70-kDa group of heat-shock proteins (Hsp70) recognize a 70-kDa polypeptide in the beta-adaptin-containing fractions. Because this polypeptide only phosphorylates in the presence of triskelions we consider it to be the uncoating ATPase, which is known to aggregate upon dissociation of the CCV coat. Our results therefore indicate that zucchini CCV contain a number of phosphorylable polypeptides equivalent to the beta, mu and sigma adaptins of bovine brain. Just as in bovine brain CCV a casein-kinase-II-like activity is associated with the zucchini CCV 50/47-kDa polypeptides, further pointing to their identity as plant mu2/mu1 adaptin equivalents.