Author: Ka Cheung Tam
Tam, Ka Cheung, 2016 Distribution of STIM1 and Orai1 and associated proteins in subcellular fractions of rat liver, Flinders University, School of Medicine
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The activation of store-operated Ca2+ channels (SOCs) at the plasma membrane is evoked by a decrease in the Ca2+ concentration in the endoplasmic reticulum (ER). The pore of SOCs is formed by a multimer Orai1 polypeptides. Stromal interacting molecule 1 (STIM1), the activator of SOCs, is localised in ER. Activation of SOCs involves movement of STIM1 in the cortical ER (cER) membrane, interaction with Orai1 in the plasma membrane and opening of the channel pore. Most published results using overexpressed STIM1 and Orai1 tagged with GFP or another tag and fluorescence microscopy immunofluorescence have shown that for unstimulated liver cells and other cell types STIM1 is distributed widely in the ER and Orai1 is predominately located on the plasma membrane. Previous results from our laboratory have provided evidence that a small region of ER rather than the whole ER is required for the activation of SOCs in hepatocytes, and this region is close to plasma membrane, most likely in the cortical ER. Throughout the cell, the ER is present as either rough (ribosomes) or smooth (no ribosomes). The region of ER called cortical ER (cER) is a large network of tubule located near the plasma membrane without the attachment of ribosome, which is thought to be the region involved in protein trafficking. Taken together, these results suggest that STIM1 should predominantly be located in the smooth ER and Orai1 in the plasma membrane. Studies from other laboratories have provided evidence that other proteins, including members of the transient receptor potential family are required for either the activation or regulation of SOCs. Moreover there are many reports of proteins other than TRP which may interact with STIM1 or Orai1. Another key finding done by other researchers from our laboratory demonstrated that the steatotic liver cells, the lipid accumulation in liver cell, causes the reduction of SOCs and the reduction can be reversed by the pretreatment of protein kinase C inhibitors. We hypothesised that the development of hepatic steatotsis is related to the phosphorylation on STIM1 and Orai1. The overall aim of this study was to investigate the distribution of endogenous STIM1 and Orai1 in rat hepatocytes using subcellular fractions of liver. The specific aims were to determine the location of STIM1 and Orai1 in miscrosome, mitochondria and the plasma membrane fraction, to identify STIM1 binding proteins in the microsomal fractions, and to investigate phosphorylation of STIM1 and Orai1 on steatotic liver cells Differential centrifugation and percoll gradient centrifugation were used to collect the subcellular fractions of nuclear fractions (NF), mitochondria (MT), heavy microsomes (HM)(derived from rough ER), light microsomes (LM) (derived from smooth ER), cytosol, and the plasma membrane (PM) from rat liver. The distribution of calreticulin (ER marker), Plasma membrane Ca2+ ATPase (PMCA), STIM1 and Orai1 was determined by western blot. Immunoprecipitation combined with LC/MS or western blot were used to search the new STIM1 binding protein in heavy microsome. We employed the technique of immunoprecipitation and western blot to detect the phosphorylation of STIM1 and Orai1 from steatotic liver cells. The results indicated that most STIM1 is found at heavy microsome (85%) with 12.5 % in light microsome. The majority of Orai1 was located in heavy microsome (44%) and light microsome (27%) fractions with only small proportion in the plasma membrane fraction. No significant change in STIM1 and Orai1 distribution was observed after the treatment of phenylephrine to deplete Ca2+ by activating α1-adrenergic receptor to induce activation of SOCs. The results of LC/MS and immunoprecipitation and western blot provides evidence that in heavy microsome STIM1 binds to peroxiredoxin 4. Several strategies were employed to test for the phosphorylation of STIM1 and Orai1 in steatotic liver cells. However, under the conditions of the experiments, no evidence for phosphorylation was obtained. Difficulties in using anti-phosphoserine antibodies and future alternative strategies to detect phosphorylated STIM1and Orai1 are discussed. It is concluded that the distribution of endogenous STIM1 and Orai1 as revealed by subcellular fractionation has some important differences to that revealed using overexpressed tagged proteins: STIM1 is localised in the rough ER as well as the smooth ER and most likely has functions in the rough ER unrelated to activation of SOCE; STIM1binds to peroxiredoxin 4 in the rough ER and this may be part of a mechanism by which STIM regulates oxidation reactions or vice versa; the localisation of Orai1 in the microsomal fraction may reflect the role of Orai1 in regulating Ca2+ in secretory granules as reported by others.
Keywords: Store- operated Ca2+ channels, STIM1, Orai1, STIM1 binding proteins, Peroxiredoxine 4, Cortical endoplasmic reticulum, subcellular fractionation, Immunoprecipitation
Subject: Medical Biochemistry thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Greg Barritt