Gilbert A. Keller

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C‐terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.

Design, construction, and in vitro analyses of multivalent antibodies.

Some Abs are more efficacious after being cross-linked to form dimers or multimers, presumably as a result of binding to and clustering more surface target to either amplify or diversify cellular signaling. To improve the therapeutic potency of these types of Abs, we designed and generated Abs that express tandem Fab repeats with the aim of mimicking cross-linked Abs. The versatile design of the system enables the creation of a series of multivalent human IgG Ab forms including tetravalent IgG1, tetravalent F(ab′)2, and linear Fab multimers with either three or four consecutively linked Fabs. The multimerized Abs target the cell surface receptors HER2, death receptor 5, and CD20, and are more efficacious than their parent mAbs in triggering antitumor cellular responses, indicating they could be useful both as reagents for study as well as novel therapeutics.

Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1‐GFP and PTS2‐GFP to the cytoplasm but did incorporate IPMP‐GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation‐competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically‐oriented C3HC4 zinc binding domain that is essential for its biological activity.

DEFECTIVE ENDOGENOUS RETROVIRUSLIKE PARTICLES OF CHINESE HAMSTER OVARY CELLS

ABSTRACT The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. We have used a high-capacity, continuous-flow, ultracentrifuge rotor to concentrate extracellular particles from culture fluid of recombinant CHO cells for molecular characterization. Equilibrium sucrose gradient sedimentation of particle preparations results in co-fractionation of reverse transcriptase activity and mammalian C-type retrovirus core proteins at a density characteristic of retrovirus particles. Electron microscope examination of gradient fractions at this density revealed the presence of particles with morphology and size similar to other characterized retroviruses. Several cDNA clones of particle RNA homologous to the endonuclease region of Moloney murine leukemia virus have been isolated and sequenced. None exhibit open reading frames capable of encoding an intact endonuclease, providing one possible explanation for the non-infectious nature of the observed particles. The presence of conserved C-type provirus sequences in the DNA of all CHO cell lines examined, as well as in Chinese hamster liver DNA, suggests that the observed particles are the products of endogenous retroviruslike elements present in the germline of Chinese hamsters. Keywords: CHO cells, C-type particles, intracisternal A-particles, endogenous retrovirus, endonuclease, reverse transcriptase, core protein, cDNA, continuous-flow ultracentrifugation, sucrose gradients.