David S. Goldfarb
University of Rochester
Department of Biology
River Campus Box 270211
Rochester, New York 14627-0211
Hutchison 436 (office)
Hutchison 435 (lab)
(585) 275-3890 (office)
(585) 275-3877 (lab)
We are interested in the molecular mechanisms that control the exchange of molecules and information between the nucleus and cytoplasm.
The molecular mechanism of translocation through the nuclear pore complex.
Nuclear transport is essential for the biogenesis of the nucleus, for housekeeping functions such as transcription and ribosome assembly, and for the regulation of gene expression during the cell cycle, in development, and in response to changing environments. Nuclear transport can be divided into two phases, targeting and translocation. Although virtually all of the protein subunits (nucleoporins) of the yeast nuclear pore complex are known, the basic mechanism of vectorial translocation remains unsolved. We discovered that two nucleoporins, Nup188p and Nup170p, control the diameter of the central diffusion channel. The diameter of the diffusion channel is significantly larger in cells lacking Nup188p or Nup170p (Fig. 1). Because Nup170p is also a docking site for translocation complexes, we are currently testing the hypothesis that Nup170p and Nup188p are directly involved in signal-directed pore gating.
The Drosophila importin/karyopherin α gene family.
Importin α is the cytosolic receptor for classical nuclear localization signals. Whereas yeasts and plants contain only importin α1 genes, animals contain three conserved clades, α1, α2, and α3 (Fig. 2). The reason animals contain three different types of importin α genes is the focus of our research. We cloned and mapped the three importin α genes from Drosophila melanogaster in order to exploit the fruit fly's facile genetics and well understood programs of development and differentiation. Interestingly, deletion of the Drosophilaimportin α2 gene causes infertility in otherwise normal flies. The full battery of Drosophilamolecular and genetic methods are being brought to bear on this fundamental animal cell biology problem.
Piecemeal microautophagy of the nucleus in S. cerevisiae.
We have discovered a novel form of autophagy (homeostatic "self-eating") that transports nuclear components directly to the vacuole for degradation and recycling in viable cells. The nucleus had not previously been considered a target for autophagy because it is an essential organelle. We now report that the S. cerevisiae nucleus is subject to Piecemeal Microautophagy of the Nucleus (PMN). During PMN, teardrop-like portions of the nucleus called blebs are pinched from the nucleus into the vacuole lumen where they are degraded (Fig. 3). The piecemeal feature of PMN allows for the selective degradation of nonessential nuclear components while preserving essential components such as chromatin, which is selectively excluded from blebs. PMN occurs at Nucleus-Vacuole (NV) junctions, which are formed through physical interactions between Vac8p in the vacuole membrane and Nvj1p in the nuclear envelope. Current work is aimed at defining the components, cargo, regulation, and molecular mechanism of PMN.
Figure 1: The nuclear pore complex diffusion channel is larger in nup170-Δ cells. A nuclear localization signal-green fluorescent protein reporter (NLS-GFP) is mostly nuclear in both wild type and nup170-Δ cells at 23oC. However, at 0oC, which inhibits active import but allows passive diffusion to continue, NLS-GFP more rapidly equilibrates across the nuclear envelope of nup170-Δ cells than wild type cells.
Figure 2: Phylogeny of the importin αs. Bootstrap values are represented as a percentage of 1000 replications. Importin α2 and α3 genes likely arose from an a1 progenitor(s). Species, protein identification numbers (PID#), and proposed α1, α2, and α3 nomenclature for conventional genes are indicated. Vertebrate genes are shown in brown, invertebrates in red, plants in green, and fungi in blue.
Figure 3: A model for PMN based on observed structures. (Stage I) NV junctions are formed by the interaction and clustering of Nvj1p (green circles) in the nuclear envelope and Vac8p (red squares) in the vacuolar membrane. (N = nucleus, V = vacuole). The formation of a nuclear bulge (Stage II) is followed by the formation of tethered blebs (Stage III). Bulging and blebbing could be considered to be early and late steps of the same stage. An intravacuolar vesicle is formed by scission of the three bleb membranes and the release of an intravacuolar vesicle into the vacuole lumen (Stage IV). Finally the PMN vesicle and its contents are degraded by vacuolar hydrolases (Stage V).
- 2006. Structure and function of nucleus-vacuole junctions: Outer nuclear membrane targeting of Nvj1p and a role in the control of tryptophan transport. J. Cell Sci. 119: 3622-3633.
- 2005. Targeting of Tsc13p to nucleus-vacuole junctions: A role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles. Molec. Biol. Cell 16: 3987-3998.
- 2005. Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death. FEMS Yeast Res. 5: 1237-51.
- 2004. Minimal nuclear pore complexes: Genomic strategy defines FG repeat domains essential for transport. Nature Cell Biol. 6: 197-206.
- 2004. Nvj1p is the outer nuclear membrane receptor for oxysterol binding protein Osh1p in Saccharomyces cerevisiae. J. Cell Sci. 117: 4959-4968.
- 2004. Importin alpha: a multipurpose nuclear transport receptor. Trends Cell Biol. 14: 505-514.
- 2004. A novel assay for replicative lifespan in Saccharomyces cerevisiae. FEMS Yeast Res. 5: 169-178.
- 2004. Minimal nuclear pore complexes: Genomic strategy defines FG repeat domains essential for transport. Nature Cell. Biol. 6: 197-206.
- 2003. Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating. Mol. Cell. Biol. 23: 534-542.
- 2003. The Drosophila melanogaster importin a3 locus encodes a essential gene required for the development of both larval and adult tissues. Genetics 165: 1943-1958.
- 2003. Piecemeal microautophagy of the nucleus in yeast. Mol. Biol. Cell. 14: 129-141.
- 2002. Drosophila melanogaster importin a1 and a3 can replace importin a2 during spermatogenesis but not oogenesis. Genetics 161: 157-170.
- 2000. Yeast nucleoporins involved in passive nuclear envelope permeability. J. Cell Biol. 149: 1027-1038.
- 2000. Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p. Mol. Biol. Cell. 11: 2445-2457.