A novel secreted form of immune suppressor factor with high homology to vacuolar ATPases identified by a forward genetic approach of functional screening based on cell proliferation

In the search for stromal-derived growth factors, we have identified a nove l secreted short form of immune suppressor factor (ISF) using a combination of a genetic approach and retrovirus-mediated functional screening. This p rotein, which we termed ShIF, was isolated based on its ability to support proliferation of a mutant clone S21, which was established from Ba/F3 cells that are usually interleukin 3-dependent but became dependent on a stroma cell line ST2 after chemical mutagenesis. ISF, a membrane protein harboring six transmembrane domains, was reported to have immunosuppressive function s. The coding region of ShIF started from the third transmembrane domain of ISF. Biochemical analysis demonstrated that ShIF was expressed in both the secreted and membrane-bound forms of 27-kDa protein, which was supposed to have an internal ATG present in the third transmembrane domain of ISF as a start codon, In addition to the full-length form of ISF, a major protein w ith a molecular size of 27 kDa was also expressed through the proteolytic p rocess of ISF, ShIF resembles this naturally occurring short form of ISF (s ISF). Deletion analysis of the major domains of ISF cDNA revealed that ShIF is an active functional domain of ISF with a capability to support prolife ration of S21 cells. Enforced expression of ShIF in MS10 cells, bone marrow stroma cells that do not express endogenous ShIF or ISF, conferred on the cells an ability to support the growth of S21 cells as well as bone marrow cells. Interestingly, ShIF shows a high sequence homology to the C-terminal part of a 95-kDa yeast vacuolar H (+)-ATPase subunit, Vph1p (39%), and a 1 16-kDa proton pump (VPP1) (54%) of the rat and bovine synaptic vesicle. The refore, it is possible that ShIF also acts as a proton pump and somehow pre vents the cells from undergoing apoptosis.