Biomedical subjects

C Feldherr

Publications and source records attributed to C Feldherr.

4 recordsLinked to original sources

PubMed2001

NTF2 monomer-dimer equilibrium.

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 microM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.

Amino Acid Substitution↗

PubMed1998

The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis.

Previtellogenic, stage-1 Xenopus oocytes produce mainly 5S and tRNA, whereas vitellogenic oocytes, stages 2-6, synthesize predominantly 18S and 28S rRNA. Using nucleoplasmin-coated gold as a transport substrate, it was determined that the shift in synthesis from small to large RNAs during oogenesis is accompanied by an increase in both the rates of signal-mediated nuclear import and the functional size of nuclear pores. It was observed that, despite the reduction in transport capacity, gold still accumulated at the cytoplasmic surface of the pores in stage-1 oocytes. This suggested that transport in these cells is limited by translocation factors, rather than by cytoplasmic binding factors. Analysis of extracts prepared from stage-1 and vitellogenic oocytes revealed that the transport factor p10 is more abundant in stage-1 cells. Microinjection of purified p10 into stage-2 oocytes reduced the nuclear import of large gold particles to the level observed in stage-1 cells. It is concluded that p10 can modulate transport through the pores by regulating the functional size of the central transporter element.

Animals↗

PubMed1995

Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

3T3 Cells↗

PubMed1994

The effects of SV40 large T antigen and p53 on nuclear transport capacity in BALB/c 3T3 cells.

In a previous study, it was found that the microinjection of purified SV40 large T antigen into the cytoplasm of BALB/c 3T3 cells significantly increased both the relative rate of signal-mediated nuclear transport and the functional size of the transport channels that are located within the pores. In this investigation, a series of mutants were employed to identify the region of large T responsible for the permeability increase. Plasmids encoding wild-type or mutant forms of large T were injected into the nucleoplasm of proliferating BALB/c 3T3 cells, and the nuclear import of nucleoplasmin-coated gold particles was analyzed approximately 18 h later. The large T mutants that were not effective in inducing the increase in nuclear transport capacity were also unable to bind p53. Further evidence that transport activity and p53 binding localize to the same region of large T was obtained by simultaneously injecting plasmids that overexpress wild-type or mutant p53 and plasmids that encode active forms of large T. It was found that wild-type p53 prevented the large T-induced transport increase; however, mutant p53, which is unable to bind to large T, had no effect. Decreasing the concentration of endogenous p53 in cells that do not contain large T, by injecting anti-p53 antibodies or plasmids that express mutant p53, resulted in a significant increase in the nuclear import of nucleoplasmin-coated gold. The latter results suggest that p53 might normally act as a transport suppressor.

3T3 Cells↗