Subcellular redistribution of Pit-2 P(i) transporter/amphotropic leukemia virus (A-MuLV) receptor in A-MuLV-infected NIH 3T3 fibroblasts: involvement in superinfection interference.
Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing epsilon epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of epsilon-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent P(i) uptake. This increase in P(i) uptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or P(i) transporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both epsilon-epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV-Pit-2 interactions.
Jobbagy
Z
Z
Laboratory of Cellular Oncology, National Institutes of Health, Bethesda, Maryland 20892, USA.
Garfield
S
S
Baptiste
L
L
Eiden
M V
MV
Anderson
W B
WB
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018985:Epitopes, B-Lymphocyte / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D028061:Phosphate Transport Proteins; D010710:Phosphates / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics; D051744:Protein Kinase C-epsilon; D011817:Rabbits; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D013347:Subcellular Fractions; D027981:Symporters; D014168:Transferrin / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism
D000199:Actins; D015415:Biomarkers; D002352:Carrier Proteins; D018985:Epitopes, B-Lymphocyte; D007527:Isoenzymes; D008164:Luminescent Proteins; D028061:Phosphate Transport Proteins; D010710:Phosphates; D011991:Receptors, Virus; D015682:Retroviridae Proteins, Oncogenic; C493589:SLC20A2 protein, human; C493587:Slc20a1 protein, mouse; C493565:Slc20a2 protein, mouse; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters; D014168:Transferrin; D014759:Viral Envelope Proteins; C067981:leukemia virus receptor, gibbon ape; D049452:Green Fluorescent Proteins; C497256:Prkce protein, mouse; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon