Replacement of the phospholipid-anchor in the contact site A glycoprotein of D. discoideum by a transmembrane region does not impede cell adhesion but reduces residence time on the cell surface

Abstract

Abstract. The contact site A (csA) glycoprotein of Dictyostelium discoideum, a cell adhesion molecule expressed in aggregating cells, is inserted into the plasma membrane by a ceramide-based phospholipid (PL) anchor. A carboxyterminal sequence of 25 amino acids of the primary csA translation product proved to contain the signal required for PL modification. CsA is known to be responsible for rapid, EDTA-resistant cohesion of cells in agitated suspensions. To investigate the role of the PL modification of this protein, the anchor was replaced by the transmembrane region and short cytoplasmic tail of another plasma membrane protein of D. discoideum. In cells transformed with appropriate vectors, PL-anchored or transmembrane csA was expressed under the control of an actin promoter during growth and development. The transmembrane form enabled the cells to agglutinate in the presence of shear forces, similar to the PLanchored wild-type form. However, the transmembrane form was much more rapidly internalized and degraded. In comparison to other cell-surface glycoproteins of D. discoideum the internalization rate of the PL-anchored csA was extremely slow, most likely because of its exclusion from the clathrin-mediated pathway of pinocytosis. Thus, our results indicate that the phospholipid modification is not essential for the csA-mediated fast type of cell adhesion but guarantees long persistence of the protein on the cell surface.

Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

“…Clathrin heavy chain is essential for internalization of plasma membrane dajumin-GFP Internalization of dajumin-GFP from the plasma membrane was confirmed using a biotin internalization assay (Barth et al, 1994) (Fig. 6A).…”

“…The assay was performed as described in Barth et al (Barth et al, 1994), 5610 6 cells were used per time point. After the cell surface reduction step, cells were lysed in 50-100 ml of lysis buffer and total protein concentration measured.…”

Acidic clusters target transmembrane proteins to the contractile vacuole inDictyosteliumcells

“…This suggests the existence of a highly specific intracellular sorting machinery involved in targeting to the CV, and of targeting signals responsible for the localization of Rh50 in the CV. To determine which domain is responsible for the localization of Rh50 on CV, we expressed a fusion protein composed of the contact site A (CsA) extracellular domain, the transmembrane domain of the integral membrane protein P29F8 (Barth et al, 1994) and the 91 residues forming the Rh50 C-terminal cytoplasmic domain (Fig. 1A).…”

“…The cDNA encoding the contact site A (CsA) protein with the transmembrane domain of the integral protein P29F8 (Barth et al, 1994) in the expression vector pDCEV4 was kindly provided by G. Gerisch (Max-Planck-Institut für Biochemie, Martinsried, Germany). The sequence of the cytoplasmic domain of the CsA-Stop construct is KTRVSQNSG.…”

Measuring Cell Adhesion Forces with the Atomic Force Microscope at the Molecular Level

“…10 6 cells/ml. For transformants HTC1 [Barth et al, 1994], CPH [Beug et al, 1973a] and T10 [Faix et al, 1992], 20 Ìg/ml of the selection marker G418 was added to stabilize csA expression. Before we took measurements on these cells, we washed and resuspended them in 17 mM K/Na buffer, pH 6.0.…”