LAC1 Temperature Sensitivity and the LIP1 Protein in Early G1 Events in

Saccharomyces cerevisiae.
1 1 2 2
Dominic Elijah DiSiliva , Jasmine Vargas , Doug R Kellogg PhD , Aparna Sreenivasan PhD
1
School of Natural Sciences, California State University, Monterey Bay
2
Molecular, Cell, & Developmental Biology, University of California, Santa Cruz

Introduction Methods Results

Cancer is viewed as the uncontrollable overproduction of cells, but it is also associated with size
abnormalities and developmental defects in the cell cycle (Pal et. al 2008). The cell cycle is split
Flowchart of Methods for LAC1 & LIP1 Investigations Successful Amplification of URA gene with LIP1 Ends
into G1, S, G2, and Mitosis. G1 and G2 phase are both gap phases in the cell cycle that allow
the cell to grow. Cellular development and size control, in early G1 events of the cell cycle, are
largely unknown. In Saccharomyces cerevisiae the genome and constituents of the cell cycle are
highly conserved, making this model organism excellent for this type of research (Harvey et. al
2003). Learning how yeast cells acquire size abnormalities during development can directly
influence our understanding about cancer and abnormal cellular development in people (Harvey
et. al 2003, McCusker et. al 2007, Anastasia et. al 2012). A conserved signaling network is
thought to control cell growth in all eukaryotic cells, but little is known about how all of the
different mechanisms of cell growth interact with each other.

Cell Cycle of S. cerevisiae

Figure 5. 1% agarose gel representing the successful amplification of the URA

gene with LIP1 flanking ends. The URA gene was amplified off the plasmid
PAG60 using primers that included 40 bp of the LIP1 gene on each end. Each
band represents 10 ul of a 100 ul reaction which were then concentrated,
purified and used for transformation into wildtype yeast.

Successful Transformation of URA into S. cerevisiae

A) B)

Figure 1. The cell cycle of Saccharomyces cerevisiae. Figure 3. A) Flowchart of methods to assess the effects of mutations in the Lac1 regions in mutant strains. B)
Flowchart of methods to assess the effects of LIP1 deletion.
The TORC2 network modulates ceramide lipid synthesis and failure to regulate ceramide activity,
in response to nutrients, results in abnormal cell size and growth. Previous studies have shown
A) Primers were designed to amplify the LAC1 gene via the Polymerase Chain Reaction
that deletion of the paralogs LAC1 and LAG1, that are encoded by ceramide synthase, result in a
(PCR). After successful amplification and confirmation of the LAC1 gene, we will then
large reduction in cell size (Lucena et al. 2018). Sphingolipid synthesis, which involves LAC1 and
commence with the generation of LAC1 temperature sensitive (TS) mutants. After Figure 5. A) Control plate for transformation process. B) Experimental plate
LAG1, is thought to be controlled by the phosphorylation and negative feedback signaling of the
generation of the LAC1 TS mutants we will perform cell cycle time course experiments in showing successful transformation of URA into S. cerevisiae wildtype strain DK186
TORC2 network (Lucena et al. 2018). Since LAG1 and LAC1 are redundant we are generating a
order to visualize the effect on cell size and growth. After the time course experiments we
series of LAC1 temperature sensitive mutations in a 𝝙lag1 strain to investigate how inactivation of
the ceramide synthase genes affect the major proteins that have been implicated in the cell size
will use immunofluorescence to visualize the effects within the cell. The effect on the Δlip1 Strains
proteins and kinases within the cell will also be visualized with western blotting and protein
checkpoint.
profiling.
In addition to our LAC1 investigation, we are investigating the role of the Lip1 protein in relation to
B) Using protocols designed by Longtine et al., we designed long primers with flanking ends of
ceramide synthesis and cell size checkpoints. Just like LAC1 & LAG1; the Lip1 protein is localized
LIP1. Then, using the primers we amplified the URA gene off the plasmid PAG60. This
within the endoplasmic reticulum and is required for ceramide synthesis (Vallee et al. 2005).
PCR product had forty bases on either side of the LIP1 sequence. Next we purified the
Recently it was discovered that the Lip1 protein forms a heteromeric complex with the LAC1 &
PCR products and concentrated them for transformation into wild type yeast. By having the
LAG1 proteins (Vallee et al. 2005). In our investigation we will observe the effect on cell size when
LIP1 base pairs attached to the URA gene we are hoping to delete the LIP1 gene through
we remove the Lip1 gene from wild type yeast using homologous recombination.
homologous recombination. The URA gene will replace the LIP1 gene and we will be able
Diagram of the TORC2 Network to visualize this by plating the transformants onto URA- media plates.

Amplification of URA with LIP1 Flanking Ends & Recombination

of the URA and LIP1 Genes. Figure 5. Δlip1 yeast, plated on -URA plates. We plated the transformants onto
-URA media to show the successful transformation of URA into wildtype yeast.

Future Work
We are currently working on the generation of LAC1 TS mutants and are now going to study
the effects of deleting LIP1 from wildtype yeast.

References
Anastasia, Steph D., et al. "A link between mitotic entry and membrane growth suggests a novel model for cell size control." J Cell Biol 197.1 (2012): 89-104.
L. Dirick, T. Bohn, K. Nasmyth. “Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae.” The Embo Journal 14.19
(1995):4803-4813.
Harvey, Stacy L., et al. "Cdk1-dependent regulation of the mitotic inhibitor Wee1." Cell 122.3 (2005): 407-420.
Figure 2. The TORC2 network proposed by the Kellogg Lab depicting the key mechanisms that govern growth rate and Harvey, Stacy L., and Douglas R. Kellogg. "Conservation of mechanisms controlling entry into mitosis: budding yeast wee1 delays entry into mitosis and is required for cell size
control." Current biology 13.4 (2003): 264-275. Harvey, Stacy L., et al. "A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast." Molecular
cell size. With the addition of the Lip1 gene. biology of the cell 22.19 (2011): 3595-3608.
McCusker, Derek, et al. "Cdk1 coordinates cell-surface growth with the cell cycle." Nature cell biology 9.5 (2007): 506.
Question: What effect do temperature sensitive mutations in LAC1 specific regions have on the Vallée B, Riezman H. Lip1p: a novel subunit of acyl-CoA ceramide synthase. The EMBO Journal. 2005;24(4):730–741.

protein kinases implicated in the cell size checkpoint in Saccharomyces cerevisiae (budding
yeast)? And how is Lip1 involved in the cell size checkpoint?
Hypotheses:
1) Conditional alleles of LAC1 will result in smaller cells
2) Conditional alleles of LAC1 will result in disruption of the activity of a number of proteins that
have been implicated in the cell size checkpoint like PP2ARTS1, SWE1, and Mss4.
3) Deletion of LIP1 will result in smaller cells.
.
Acknowledgments
Much appreciation goes to Dr. Aparna Sreenivasan for her support and mentoring, to Dr. Doug
Kellogg for his collaboration, and to the Undergraduate Research Opportunities Center (UROC)
Figure 4. A) Diagram of URA being amplified with LIP1 flanking ends. B) Deletion of LIP1 via recombination of for their generous contribution. I also want to thank Leah Martin and Alexander Ferreira for their
the URA gene with LIP1 flanking ends. previous work on the yeast strains.