Regulation of constitutive protein transit by phospholipase D in HT29-cl19A cells.
Phospholipase D (PLD) plays a central role in the control of vesicle budding and protein transit. We previously showed that in resting epithelial HT29-cl19A cells, PLD is implicated in the control of constitutive protein transit, from the trans-Golgi network to the plasma membrane, and that phorbol ester stimulation of protein transit is correlated with PLD activation (Auger, R., Robin, P., Camier, B., Vial, G., Rossignol, B., Tenu, J.-P., and Raymond, M.-N. (1999) J. Biol. Chem. 274, 28652-28659). In this paper we demonstrate that: 1) PLD is not implicated in the earliest phases of protein transit; 2) PLD controls apical but not basolateral protein transit; 3) HT29-cl19A cells express PLD1b and PLD2a mRNAs and proteins; 4) the expression of a catalytically inactive mutant of PLD2 (mPLD2-K758R) significantly inhibited apical constitutive protein transit whereas expression of a catalytically inactive mutant of PLD1 (hPLD1b-K898R) prevented increases in the rate of apical transit as triggered by phorbol esters; 5) PLD2 appears to be located in a perinuclear region containing the Golgi whereas PLD1, which is scattered in the cytoplasm in resting cells, is translocated to the plasma membrane after phorbol ester stimulation. Taken together, these data lead to the conclusion that in HT29-cl19A cells, both PLDs regulate protein transit between the trans-Golgi network and the apical plasma membrane, but that they do so at different steps in the pathway.
Denmat-Ouisse
L A
LA
Laboratoire de Biochimie des Transports Cellulaires, CNRS, U.M.R. 8619, bâtiment 430, Université Paris XI, 91405 Orsay Cedex, France.
Phebidias
C
C
Honkavaara
P
P
Robin
P
P
Geny
B
B
Min
do S
DS
Bourgoin
S
S
Frohman
M A
MA
Raymond
M N
MN
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms; D021381:Protein Transport / Q000502:physiology*; D011868:Radioisotopes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014361:Trypsin Inhibitors / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D020033:Protein Isoforms; D011868:Radioisotopes; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D000431:Ethanol; D010739:Phospholipase D