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The prenylated Rab acceptor (PRA) 1 is a protein that binds prenylated Rab GTPases and inhibits their removal from the membrane by GDI. We describe here the isolation of a second isoform that can also bind Rab GTPases in a guanine nucleotide-independent manner. The two PRA isoforms showed distinct intracellular localization with PRA1 localized primarily to the Golgi complex and PRA2 to the endoplasmic reticulum (ER) compartment. The localization signal was mapped to the COOH-terminal domain of the two proteins. A DXEE motif served to target PRA1 to the Golgi. Mutation of any one of the acidic residues within this motif resulted in significant retention of PRA1 in the ER compartment. Moreover, the introduction of a di-acidic motif to the COOH-terminal domain of PRA2 resulted in partial localization to the Golgi complex. The domain responsible for ER localization of PRA2 was also confined to the carboxyl terminus. Our results showed that these sorting signals were primarily responsible for the differential localization of the two PRA isoforms.

Background Eukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network’s dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation. Results The model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes. Conclusions This study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes.

The origin recognition complex (ORC) is essential as a scaffold for the assembly of prereplicative complexes (pre-RCs) in G(1) phase of the cell cycle. Some models have proposed that once origins have been licensed for DNA replication, ORC is dispensable for MCM protein association, and ensuing DNA replication. Although budding yeast Orc6 is not needed for origin recognition or binding in vitro, we have recently shown that this ORC subunit is required in late G(1) phase for maintenance of MCMs, and subsequent DNA replication. Further investigation shows that depletion of Orc6 results in displacement of MCM proteins from both early- and late-firing origins, and eventually results in the activation of the Rad53 checkpoint kinase, consistent with incomplete DNA replication. Loss of MCM association at origins may be mediated by the displacement of Mcm10 and/or Orc2 as a consequence of late G(1) Orc6 depletion.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G 1 phase assembly of pre‐replicative complexes (pre‐RC). Only the Orc1‐5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6‐YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre‐RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G 1 , Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.

Rab is a family of small Ras-like GTPases regulating intracellular vesicle transport. We have previously reported that prenylated Rab acceptor or PRA1 interacts with Rab GTPases and vesicle-associated membrane protein (VAMP2). Structural prediction programs suggest that PRA1, with its two extensive hydrophobic domains, is likely to be an integral membrane protein. However, subcellular fractionation and immunocytochemical analyses indicated that PRA1 is localized both in the cytosol and tightly associated with the membrane compartment. The membrane-bound form can be partially extracted with physiological buffer and urea, suggesting that PRA1 is an extrinsic membrane protein. Deletion of the carboxyl-terminal domain resulted in a protein that behaved as an integral membrane protein, indicating that this domain plays an essential role in maintaining PRA1 in a soluble state. PRA1 can also bind weakly to GDP dissociation inhibitor (GDI), a protein involved in the solubilization of membrane-bound Rab GTPases. Addition of PRA1 inhibited the extraction of membrane-bound Rab3A by GDI, suggesting that membrane localization of Rab GTPases is dependent on the opposing action of PRA1 and GDI. The binding of Rab and VAMP2 to PRA1 is mutually exclusive such that Rab3A can displace VAMP2 in a preformed VAMP2-PRA1 complex.