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The dynamic nature of the Golgi apparatus in biosynthesis and structure

The Golgi apparatus in mammalian cells consists of several flat cisternae surrounded by individual membranes. Still, this organelle system is regarded as one functional unit for modification/processing of proteins and lipids in the secretory pathway, underway to endosomes and the plasma membrane. The Golgi apparatus is flexible both in size and function and tends to have a larger volume in cell types with a higher flow in the secretory route. The content of processing enzymes, like glycosyl- and sulfotransferases, and transporter proteins in the Golgi membrane determines the output of glycoproteins and proteoglycans. These proteins organize in functional domains in Golgi cisternae.

We have over-expressed a transporter protein (PAPST-1) that translocates PAPS (3'-phosphoadenosine-5'-phosphosulfate) from the cytoplasm against a concentration gradient into the lumen of the Golgi apparatus. This leads to a large enhancement of a band defined as the Golgi fraction after subcellular fractionation of epithelial MDCK cells. This fraction will be subjected to proteomic analysis by mass spectrometry (MS) in collaboration with the Thiede-group (BMB), to address which proteins that are co-induced with PAPST-1.

In parallel, the group of Steven Wilson (Department of Chemistry) has, in collaboration with the Prydz-group, developed an assay based on HPLC and MS, to quantify the amount/concentration of PAPS in biological samples, like cell lysates and Golgi fractions. By manipulating the supply of PAPS available to proteoglycan synthesis in the Golgi apparatus lumen, we can address how epithelial MDCK cells give priority to certain sulfotransferases. To estimate the PAPS concentration in different biochemical and cellular situations, we aim at determining the 3D-structure of the Golgi apparatus to calculate the average Golgi volume and combine the data with those from the HPLC/MS analysis. The 3D-analysis will be performed with confocal microscopy and will be supervised by Frode Skjeldal.

We have ongoing work where we try to regulate the expression of PAPST-1 and a related protein by CRISPR/CAS technology. If these attempts succeed, we will pursue this experimental line further in our studies of the dynamics of the Golgi apparatus.

Relevant references

  1. Dick, G., Gr?ndahl, F., and Prydz, K. 2008. Overexpression of the PAPS transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells. Glycobiology 18; 53-.
  2. Dick, G., Akslen-Hoel, L.K., Gr?ndahl, F., Kjos, I., Maccarana, M., and Prydz, K. 2015. PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells. Glycobiology 25; 30-.
  3. Fjeldstad, K., Pedersen, M.E., Vuong, T.T., Kolset, S.O., Nordstrand, L.M., and Prydz, K. 2002. Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes. J. Biol. Chem. 39; 36272-.
  4. Safayian, F., Kolset, S.O., Prydz, K., Gottfrisson, E., Lindahl, U., and Salmivirta, M. 1999. Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate. J. Biol. Chem. 274; 36267-
  5. Kreuger, J., Prydz, K., Pettersson, R.F., Lindahl, U., and Salmivirta, M. 1999. Characterization of fibroblast growth factor 1 binding heparan sulfate domain. Glycobiology 9; 723-.
  6. Prydz, K. and K.T. Dalen. 2000. Synthesis and sorting of proteoglycans. J. Cell Sci. 113; 193-.
  7. Prydz, K.2015. Determinants of glycosaminoglycan (GAG) structure. Biomolecules 5; 2003-.
Published Mar. 22, 2018 10:28 AM - Last modified Apr. 19, 2018 8:14 AM

Supervisor(s)

Scope (credits)

60

One or two students may be affiliated with the project.