A novel NH2-terminal, nonhydrophobic motif targets a male germ cell-specific hexokinase to the endoplasmic reticulum and plasma membrane

Although three germ cell-specific transcripts of type 1 hexokinase exist in murine male germ cells, only one form, HK1-sc, is found at the protein lev el. This single isoform localizes to three distinct structures in mouse spe rmatozoa: the membranes of the head, the mitochondria in the midpiece, and the fibrous sheath in the flagellum (Travis, A. J., Foster, J. A. Rosenbaum , N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998 ) Mol. Biol, Cell 9, 263-276), The mechanism by which one protein is target ed to multiple sites within this highly polarized cell poses important ques tions of protein targeting. Because the study of protein targeting in germ cells is hampered by the lack of established cell lines in culture, constru cts containing different domains of the germ cell-specific hexokinase trans cripts were linked to a green fluorescent protein and transfected into hexo kinase-deficient M+R42 cells. Constructs containing a nonhydrophobic, germ cell-specific domain, present at the amino terminus of the HK1-SC protein, were targeted to the endoplasmic reticulum and the plasma membrane. Mutatio nal analysis of this domain demonstrated that a complex motif, PKIRP-PLTE ( with essential residues italicized), represented a novel endoplasmic reticu lum-targeting motif, Constructs based on another germ cell-specific hexokin ase transcript, HK1-sa, demonstrated the specific proteolytic removal of an amino-terminal domain, resulting in a protein product identical to HK1-SC, Such processing might constitute a regulatory mechanism governing the spat ial and/or temporal expression of the protein.