Abstract
The proton-translocating vacuolar ATPase (V-ATPase) acidifies the endocytic network of eukaryotic cells. Although all eukaryotic cell types require low to moderate levels of V-ATPase, some proton-secreting cells express amplified levels for use in specialized membrane domains. To characterize genetic elements required for this heightened expression, we studied transcription and stability of mRNA encoding the V-ATPase c subunit in a low expressing fibroblast cell line (NIH 3T3) and a high expressing macrophage cell line (RAW 264.7). Isolation of the promoter and mapping of the transcriptional start site indicated that the c subunit promoter is TATA-less and initiates transcription at a single site. Promoter activity was regulated through the same transcription factor binding sites in both cell types, which showed no discernible difference in rates of c subunit transcription. In contrast, c subunit transcripts showed markedly greater stability in RAW cells than in 3T3 cells, as did other constitutively expressed V-ATPase subunit transcripts. Only the B and 'a' subunits, which are expressed in multiple isoforms, were not regulated solely by mRNA stability. These results suggest that overall expression levels of the V-ATPase are set primarily by regulation of mRNA stability and that transcriptional mechanisms determine subunit composition in varying cell types.
Highlights
The proton-translocating vacuolar ATPase (VATPase) acidifies the endocytic network of eukaryotic cells
All eukaryotic cells require low to moderate levels of V-ATPase to acidify their endocytic networks, specialized, proton-secreting cells such as kidney tubule epithelia, macrophages, and osteoclasts express levels of the enzyme that are greater by an order of magnitude or more
Our previous studies suggested further that V-ATPase mRNAs are expressed in the same stoichiometry as V-ATPase subunits [22] and that increases in V-ATPase expression are mediated by coordinated up-regulation of all subunit mRNAs [22, 23]
Summary
V-ATPase, vacuolar proton-translocating ATPase; CMV, cytomegalovirus; Inr, initiator element; UTR, untranslated region. Osteoclasts and renal intercalated cells shuttle V-ATPases between intracellular stores and the plasma membrane in response to extracellular stimuli [3, 4], whereas macrophages require high levels of V-ATPase for acidification of phagocytic vacuoles [5] and regulation of intracellular pH [6, 7]. These cells and others that utilize V-ATPases for specialized purposes often express tissuerestricted isoforms of a few V-ATPase subunits. These data suggest that transcriptional control may determine the subunit content of the V-ATPase, whereas regulation of mRNA stability determines its overall expression levels
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