The molecular diversity of the luminal endothelial cell surface arising in vivo from local variations in genetic expression and tissue microenvironment may create opportunities for achieving targeted molecular imaging and therapies. Here, we describe a strategy to identify probes and their cognate antigens for targeting vascular endothelia of specific organs in vivo. We differentially screen phage libraries to select organ-targeting antibodies by using luminal endothelial cell plasma membranes isolated directly from tissue and highly enriched in natively expressed proteins exposed to the bloodstream. To obviate liver uptake of intravenously injected phage, we convert the phage-displayed antibodies into scFv-Fc fusion proteins, which then are able to rapidly target select organ(s) in vivo as visualized directly by ␥-scintigraphic whole-body imaging. Mass spectrometry helps identify the antigen targets. This comprehensive strategy provides new promise for harnessing the power of phage display for mapping vascular endothelia natively in tissue and for achieving vascular targeting of specific tissues in vivo.antibody ͉ vascular targeting ͉ endothelial cell ͉ protein expression

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After synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin ␤-importin 7 (Imp␤-Imp7) heterodimer. H1 histones contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Imp␤-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitoninpermeabilized cell import assay we show that Imp␤ is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Imp␤ and Imp7 is the functional receptor for the entire Cterminal domain. However, short fragments of the Cterminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Imp␤ with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histones. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Imp␤-Imp7 heterodimer is specialized for NLS consisting of extended basic domains.Histones are the protein component of the fundamental structural unit of eukaryotic chromatin, the nucleosome. The nucleosome is composed of a core particle consisting of a histone octamer with 146 bp of DNA bent around its surface and an approximately 50-bp linker DNA connecting two adjacent core particles. This linker DNA is associated with H1 histones; hence they are termed linker histones (1). The four core histones H2A, H2B, H3, and H4 form the histone octamer. Histones are small, very basic proteins. The linker histone H1 is highly enriched in lysine, H2A and H2B are moderately lysinerich, and H3 and H4 are rich in arginine. The size of core histones varies between 11 kDa (H4) and 15 kDa (H3), whereas the H1 histones have a size of ϳ22 kDa. In mammals the class of H1 histones, the linker histones, comprises seven different subtypes, termed H1.1-H1.5, H1°, and H1t (2). Histones are composed of three domains, the very basic, unstructured C-and N-terminal domains and the hydrophobic, central globular domain, which forms the histone fold motif in the case of the core histones (3) and is involved in histone-histone interactions. During the S phase of the cell cycle, a vast amount of newly synthesized histones has to be imported into the nucleus for the formation of nucleosomes on newly replicated DNA. Thus, histones are among the most abundant substrates for nuclear transport during the...

To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein.We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation V3-fold in wild type proteoliposomes. mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain. In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide. We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.z 1998 Federation of European Biochemical Societies.