Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment.
Folding and oligomerization of most plasma membrane glycoproteins, including those involved in ion transport, occur in the ER and are frequently required for their exit from this organelle. It is currently unknown, however, where or when in the biosynthetic pathway these proteins become functionally active. AE1 and AE2 are tissue-specific, plasma membrane anion transport proteins. Transient expression of AE2 in a eukaryotic cell line leads to an increase in stilbene inhibitable whole cell 35SO4(2-)-efflux consistent with its function as a plasma membrane anion exchanger. No such increased transport activity was observed in AE1 transfectants, despite the fact that the two proteins were synthesized in roughly equal portions. In contrast, both AE1 and AE2 expression resulted in significant increase in Cl-/SO4(2-)-exchange in crude microsomes demonstrating that both AE1 and AE2 cDNAs encode functional proteins. Immunofluorescence staining and pulse-chase labeling experiments revealed that while 60% of AE2 is processed to the cell surface of transfectants, AE1 is restricted to an intracellular compartment and never acquires mature oligosaccharides. Crude microsomes from transfected cells were fractionated into plasma membrane and ER-derived vesicles by con A affinity chromatography. All of the AE1 and approximately half of the cellular AE2 was eluted with the ER vesicles, confirming their intracellular localization. Anion transport measurements on these fractions confirmed that the ER-restricted anion exchangers were functional. We conclude that AE1 and AE2 acquire the ability to mediate anion exchange at an early stage of their biosynthesis, before their exit from the ER.
Ruetz
S
S
Department of Biological Sciences, Stanford University, California 94305-5020.
Lindsey
A E
AE
Ward
C L
CL
Kopito
R R
RR
D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism; D017920:Antiporters; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004247:DNA; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D017136:Ion Transport; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D064606:SLC4A Proteins; D013431:Sulfates / Q000378:metabolism
D027321:Anion Transport Proteins; D000838:Anions; D017920:Antiporters; D008565:Membrane Proteins; D064606:SLC4A Proteins; D013431:Sulfates; D004247:DNA