Analyzing Spatial Localization of Proteins of the Endoplasmic Reticulum in Budding Yeast, S. Cerevisiae, During Cell Division
University of Delaware
The first organelle of the secretory pathway, the endoplasmic reticulum (ER), is an essential organelle responsible for multiple critical processes of the cell. BiP, the molecular chaperone residing in the ER lumen of yeast, is involved in many of these intracellular processes including karyogamy (nuclear fusion during the mating of diploid cells), ER translocation of nascent protein, protein folding and maturation, and ERAD (ER associated degradation) of misfolded proteins. Due to BiP’s involvement in karyogamy and ER biogenesis, we have examined BiP’s role in the division of budding yeast, S. cerevisiae. The ER is a complex structure consisting of three subdomains including: (i) the peripheral ER found directly beneath the plasma membrane of the cell; (ii) the perinuclear ER, a structure that is continuous with the nuclear envelope; and (iii) a network of ER tubules connecting the former two domains. Using a combination of confocal light microscopy techniques and fluorescent protein variants, we have monitored dividing yeast cells expressing ER luminal proteins BiP and GFP retained in the ER (KGFP), ER membrane protein Sec61, and nuclear pore complex (NPC) protein Nup49, which spans both the perinuclear ER and nuclear membranes. Observation of ER inheritance during cellular division was classified as a three-step process identified by the segregation of ER subdomains. Phase 1 included the spatial localization of proteins to distinct areas of the peripheral ER as a bud emerges and continues to grow. The timescale of Phase 1 was approximately 55 minutes. Phase 2 consisted of nuclear division and was identified as the segregation of the perinuclear ER between the mother and daughter cell, which lasted for approximately 35 minutes. Phase 3 included complete separation of the mother and daughter cells. The time period of Phase 3 was approximately 18 minutes, during which ER luminal and membrane proteins returned to a homogeneous spatial distribution in all ER subdomains. We have confirmed our hypothesis that spatial heterogeneity exists among ER resident and NPC proteins during cellular division and is a consequence of protein function during ER biogenesis. Our experiments continuously monitored ER fusion proteins throughout different time points of cell division. Results include comparison of the ER luminal protein, BiP, and ER membrane protein, Sec61, which indicate that spatial heterogeneity exists in the perinuclear ER during different stages of cell division. KGFP, a construct that is not involved in any ER process, has been used as a control in order to confirm that spatial localization of ER resident proteins is dictated by the protein’s function. In addition, we have evaluated the localization of a luminal protein, BiP-mCherry, compared to the NPC identified by Nup49-Venus. Co-localization of these two proteins decreased throughout the progression of cell division, suggesting that BiP is required to leave the perinuclear ER of the mother cell during organelle biogenesis. In order to determine the mechanism which controls ER inheritance, disruption of yeast microtubules and actin cytoskeleton was performed. Actin depolymerization disrupted ER morphology and altered ER protein spatial localization in budding yeast cells, suggesting a pivotal role for actin in mother-daughter budding. Our research has offered a better understanding of how various ER resident proteins maintain distinct spatial localization patterns as a consequence of their unique functions during ER inheritance. Furthermore, we have identified specific classifications of spatial localization effects of protein in multiple subdomains of the ER and provided timescales of these specific phases based upon population studies.