Membrane topology of the Streptomyces lividans type I signal peptidases

Most bacterial membranes contain one or two type I signal peptidases (SPase s) for the removal of signal peptides from export proteins. For Streptomyce s lividans, four different type I SPases (denoted SipW, SipX, SipY, and Sip Z) were previously described. In this communication, we report the experime ntal determination of the membrane topology of these SPases. A protease pro tection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ hav e a predicted topology similar to that of SipY. These three S. lividans SPa ses are currently the only known prokaryotic-type type I SPases of gram-pos itive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains on ly the N-terminal transmembrane segment, similar to most type I SPases of g ram-positive bacteria. Functional analysis showed that the C-terminal trans membrane anchor of SipY is important to enhance the processing activity, bo th in vitro as well as in vivo. Moreover, for the S. lividans SPases, a rel ation seems to exist between the presence or absence of the C-terminal anch or and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown t o be of major importance to the cell. Accordingly, for SipW, missing the C- terminal anchor, a minor role in preprotein processing was found.