Intracellular trafficking of the vacuolar H+-ATPase accessory subunit Ac45.
Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.
Jansen
E J
EJ
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands.
Holthuis
J C
JC
McGrouther
C
C
Burbach
J P
JP
Martens
G J
GJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D004252:DNA Mutational Analysis; D004705:Endocytosis; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*; D029867:Xenopus Proteins
C089598:ATP6AP1 protein, human; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C484227:atp6ap1.1 protein, Xenopus; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases