Binding affinity and capacity of putative adaptor-mediated sorting of a Type I membrane protein in Leishmania mexicana.
The membrane-bound acid phosphatase (MBAP), a Type I membrane protein predominantly associated with endosomal/lysosomal structures of Leishmania mexicana promastigotes, contains motifs in its cytosolic COOH-terminal tail (-MEVWRRYMKFKNKQSEAIIV-COOH) akin to tyrosine- and di-leucine-based sorting signals in multicellular organisms. Here, we first show that the COOH-terminal residues IIV of MBAP, but not the Y-residue, are required for endosomal targeting, suggesting specific binding to an adaptor complex at the cell surface. We then determine whether specific binding can be saturated by analysing the efficiency of endosomal targeting for increasing numbers of MBAP molecules per cell. The ratio of the steady-state abundance of wild-type MBAP on the cell surface to MBAP on endosomes increases until the distribution is no longer different from that observed for a mutant MBAP which lacks the IIV-motif or for a glycosylphosphatidylinositol-anchored form, both of which are distributed according to bulk membrane flow. A quantitative analysis of these in vivo results indicates specific binding to a putative adaptor complex with an affinity of about 10-4M to 50,000 sorting sites on the cell surface.
Weise
Frank
F
Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, D-72076 Tübingen, Germany.
Thilo
Lutz
L
Engstler
Markus
M
Wiese
Martin
M
Benzel
Isabel
I
Kühn
Christina
C
Bühring
Hans-Jörg
HJ
Overath
Peter
P
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D011992:Endosomes / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D007425:Intracellular Membranes / Q000378:metabolism; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021382:Protein Sorting Signals; D021381:Protein Transport
D033942:Adaptor Proteins, Vesicular Transport; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D000135:Acid Phosphatase