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Development of a Novel Assay to Monitor Nuclear Pore Complex in Saccharomyces cerevisiae

  • Author(s): Stanton, Leslie Helen
  • Advisor(s): Weis, Karsten
  • et al.
Abstract

Eukaryotic cells can be distinguished from prokaryotic cells because they posses membrane bound organelles. The presence of organelles in cells allowed cellular processes to be isolated into compartments, thus allowing additional levels of regulation to be applied to simple cellular processes. One of these membrane bound organelles, the nucleus, functions to isolate the cell's DNA from the cytoplasm. Large aqueous pores span the nuclear envelope and determines which molecules can enter and exit the nucleus. These channels are called Nuclear Pore Complexes (NPCs). The NPC is made up of a central core that spans the NE, a nuclear basket structure, fibers that extend into the cytoplasm, and is composed of only ~33 different proteins called Nucleoporins. The NPC exhibits an eight-fold rotation symmetry around the plane of the NE, and nucleoporins are present in eight, or multiples of eight, copies. Although the structure and composition of the NPC is well characterized, the process in which NPCs are inserted into the intact NE of yeast is unknown.

In order to learn more about NPC assembly into the intact NE, I developed an assay to monitor the distribution of old and new nucleoporins in live S. cerevisiae cells. I used the photoconvertable fluorescent protein Dendra to examine a report that new NPC assembly occurs exclusively in daughter buds while old NPCs remain with mother cells. We examined two different Nups and observed new pore formation in both mother and daughter cells, additionally we determined that old pores are inherited by both mother and the daughter cells. We hypothesized that the differences in our observations from the previous report was due to differences experimental technique.

To begin to understand the early events of NPC assembly, we first determined which proteins interact with the essential transmembrane Nup Ndc1. We then constructed conditional mutants of different combinations of these proteins and determined that Nup59 is functionally redundant with the combined element of Pom34 and Pom152. We show that these conditional mutants are not viable and result in mislocalization of core and cytoplasmic Nups. We use the Dendra assay to show that depletion of these elements result in a reversible defect in NPC assembly.

Finally, we examine the role of Nup157 and Nup170 in NPC assembly. A nup157∆ strain conditionally expressing Nup170 results in mislocalization of core and cytoplasmic reporter Nups, but not nuclear Nups. This strain also has a reduced number of NPCs by EM in non-permissive conditions. Cells depleted of Nup170 and Nup157 were examined with the Dendra assay and new protein accumulated in the cytoplasm suggesting a block in NPC assembly. This block is overcome by reintroduction of Nup170. The work presented here represents a new way to study NPC assembly and reveals a few early events in NPC assembly.

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