b -domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu 601 -Ala 692 ) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu 601 -Ala 692 in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.

The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "␣-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "␤-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal ␣-domain and a 30-kDa C-terminal ␤-domain. We have previously shown that a recombinant form of the ␤-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31-to 39-amino-acid region found immediately upstream of the ␤-domain that was essential for surface expression. This 31-to 39-amino-acid linker region, together with the ␤-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.

Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.

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