TRANSPORT AND CELL WALL IN EUKARYOTES

Prof. R. Krämer, PD Dr. S. Waffenschmidt, M. Albertsen, I. Bellahn, K. Bollig, M.T. Finken, 
E. Glees, R. Hecker, T. Kop-Weiershausen, M. Schauen

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SCIENTIFIC BACKGROUND
In Saccharomyces cervisiae the vacuole is of crucial significan-ce for pH- and ion homeostasis, for degradation of macromole-cules, for detoxification and storage.
In Chlamydomonas reinhardtii hydroxyprolin-rich glycoproteins (HRGPs) are essential for cell wall formation and cell-cell inter-action of gametes.		 AIMS
  • The aim of the first project is the physiological, biochemical and molecular definition of vacuolar carriers for small solutes, such as amino acids, polyamines etc.
  • In the second project, we want to understand biosynthesis of HRGPs, post-translational processing, assembly and crosslin-king into a three-dimensional network.
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    The currently known vacuolar transport systems in the yeast Saccharomyces cerevisiae. The V-ATPase establishes an electrochemical proton potential across the vacuolar membrane, which is the driving force for secondary carriers, transporting ions, amino acids and polyami-nes. In addition, ATP-dependent ABC transporters are present.

    		 METHODS AND STRATEGY
    A number of putative vacuolar carriers of S. cerevisiae were assigned in silico. These open reading frames were tagged, deleted and overexpressed for localization studies and phenotype analyis. The distribution and dynamic fluxes of metabolites in the intracellular compartments of the cytoplasm and the vacuole are analyzed after selective permeabilisation by HPLC, GC and mass spectrometry in mutant and wild type cells grown under various growth and stress conditions. Transport competent vacuolar vesicles are studied with respect to solute transport and their constituent proteins are analyzed by 2D-gel electrophoresis and mass spectrometry.

    After enzymatic lysis, C. reinhardtii regenerates an intact cell wall within three hours. The regenerating cell allowed to analyze cross-linking activities. Isolated outer wall proteins are used in in vitro interaction studies, and CD-spectroscopy is used for structural characterization. Posttranslationally added sugar side chains are elucidated using GC, NMR and mass spectrometry. The function of distinct sugar moieties is analyzed by compa-ring sugar side-chains of wild type and glycosylation-defect mutants. 

    Previous investigations were aimed on structural and functional isolation of various mitochondrial carrier proteins, in particular the phosphate, the aspartate/glutamate, the ADP/ATP and the citrate carrier from beef heart and from S. cerevisiae. Functional analysis was performed in reconstituted systems.
    FUNDING
    • DFG (GK 306/2)
    • DFG (SFB 189)
    • BMBF (WTZ Tschech. Rep.)
    COOPERATION
    SELECTED PAPERS
    • Förster, C., Revuelta, J.L., Krämer, R. (2001). Carrier-mediated transport of riboflavin in Ashbya gossypii. Appl. Microbiol. Biotechnol., 55, 85-89.  [Abstract]

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    • Ferris, P.J., Woessner, J.P., Waffenschmidt, S., Kilz, S., Drees, J., Goodenough, U.W. (2001). Glycosylated polyproline II rods with kinks as structural motif in plant hydroxyproline-rich glycoproteins. Biochemistry, 40, 2978-2987. [Abstract]

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    • Waffenschmidt, S., Kusch, T., Woessner, J.P. (1999). A transglutaminase immunologically related to tissue transglutaminase catalyzes cross-linking of cell wall proteins in Chlamydomonas reinhardtii.  Plant. Physiol., 121, 1003-1015.  [Abstract]

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    • Förster, C., Santos, M., Revuelta, J.L., Krämer, R. (1998). Organismen zur extrazellulären Herstellung von Riboflavin. Deutsche Patentanmeldung 19839567.1.

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    • Förster, C., Santos, M., Ruffert, S., Krämer, R., Revuelta, J.L. (1999). Physiological consequence of disruption of the VMA1-gene in the riboflavin overproducer Ashbya gossypii. J. Biol. Chem., 274, 9442-9448. [Abstract]

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    • Schröers, A., Burkovski, A., Wohlrab, H., Krämer, R. (1998). The phosphate carrier from yeast mitochondria: dimerization as a prerequisite for function. J. Biol. Chem., 273, 14269-14276.  [Abstract]

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    • Schröers, A., Krämer, R., Wohlrab, H. (1997). The reversible antiport-uniport conversion of the phosphate carrier from yeast mitochondria depends on the presence of a single cysteine.  J. Biol. Chem., 272, 10558-10564.  [Abstract]