Filamentous Phage are Released from the Bacterial Membrane by a Two-step Mechanism Involving a Short C-terminal Fragment of pIII Jasna Rakonjac, Jian-nong Feng and Peter Model* The Rockefeller University 1230 York Avenue, New York NY 10021, USA Filamentous phage assemble at the membrane of infected cells. The phage ®lament is released from the membrane at the end of assembly, after four to ®ve copies of the minor proteins, pIII and pVI, have been added to the end of the virion. In the absence of pIII or pVI, phage ®laments are not released, but remain associated with the cells. The C- terminal portion of pIII, termed the ``C'' domain, is required for the release of stable virions. With the use of pIII C-terminal fragments of increasing size, termin- ation of assembly can be divided into various steps. An 83-residue frag- ment leads to the incorporation of pVI into the assembling phage, but does not release it from the membrane. A slightly longer fragment (93 residues) is suf®cient to release the particle into the culture supernatant. However, these released particles are unstable in the detergent, sarkosyl, which does not disrupt wild-type phage. A fragment of >121 residues is needed for the particle to become detergent resistant. Thus, the C-domain can be divided into two subdomains: C2, suf®cient for release, and C1, required for virion stability. A model for termination of phage assembly is proposed in which pIII and pVI dock to the membrane-associated ®lament and form a pre- termination complex. Then, a conformational change involving the C2 domain of pIII disrupts the hydrophobic interactions with the inner membrane, releasing the phage from the cells. The pIII-mediated release of phage from the membranes points to one possible mechanism for exci- sion of membrane-anchored protein complexes from lipid bilayers. # 1999 Academic Press Keywords: ®lamentous phage; gene III-encoded protein; gene VI-encoded protein; phage assembly *Corresponding author Introduction The entry of ®lamentous phage into their host is accompanied by the deposition of their major coat protein into the host's membrane. Virion assembly occurs at the membrane and is completed when the phage is released from the assembly site. Both entry and release are dependent on the integrity of a minor coat protein, pIII. The virion of f1 (fd, or M13) phage is 880 nm long and 6-7 nm in diameter (Model & Russel, 1988) It is comprised of a circular single-stranded DNA (ssDNA) genome, wrapped in a tube com- posed of around 2700 copies of the major coat pro- tein, pVIII. The two ends of the ®lament bear two different pairs of proteins, called minor coat pro- teins, each present in four or ®ve copies per par- ticle (Goldsmith & Konigsberg, 1977; Woolford et al., 1977; Grant et al., 1980; Lin et al., 1980). One pair (pVII and pIX) is at the end at which assembly initiates, while the other pair (pIII and pVI) is at the end of the phage at which assembly terminates (Lopez & Webster, 1983). The structural proteins of the f1 virion are very small and hydrophobic, with the exception of pIII, E-mail address of the corresponding author: model@rockvax.rockefeller.edu Abbreviations used: Amp, ampicillin; Cm, chloramphenicol; gI, gene I ; gIII, gene III ; gVI, gene VI; IPTG, isopropyl-b-D-thiogalactopyranoside; PCR, polymerase chain reaction; pIII, gene III protein; pVI, gene VI protein; pVIII, gene VIII protein; psp, phage shock protein operon; RNase A, ribonuclease A; wt, wild-type; (..), denotes plasmid-carrier state; ssDNA, single-stranded DNA. Article No. jmbi.1999.2851 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 289, 1253±1265 0022-2836/99/251253±13 $30.00/0 # 1999 Academic Press