IDENTIFICATION AND CHARACTERIZATION OF PROTEASE-RESISTANT SECA FRAGMENTS - SECA HAS 2 MEMBRANE-INTEGRAL FORMS

We have identified and characterized the protease-resistant SecA fragm ents (X. Chen, H, Xu, and P, C, Tai, J, Biol, Chem, 271:29698-29706, 1 996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis, The 66-, 36-, and 27-kDa proteol ytic fragments in the membranes all start at Met(1), whereas the 48-kD a fragment starts at Glu(361). The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from differe nt SecA molecules, These two fragments were generated differently in r esponse to ATP hydrolysis and protein translocation, Furthermore, the presence of membrane is required for the generation of the 48-kDa frag ment but not for that of the 66-kDa fragment, These data suggest that there are two different integral forms of SecA in the membrane: SecA(S ) and SecA(M), The combination of these two forms of SecA has several membrane-interacting domains. Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa Fragments could be derived fr om urea-or Na2CO3-washed membranes, Moreover, all fragments are resist ant to extraction with a high concentration of salt or with heparin, b ut the membrane-specific 48-kDa SecA domain is more sensitive to Na2CO 3 or urea extraction, This suggests that this domain may interact with other membrane proteins in an aqueous micro-environment and therefore may form a part of the protein-conducting channel.