The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, Not a SAR Domain

Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI–T complex.

Complete Genome Sequence of Rhizobium phaseoli Podophage Palo

Here, we present the genome of Palo, a T7-like podophage of Rhizobium phaseoli . The genome is 46.3 kb and contains 58 predicted protein-coding genes, including a novel signal-anchor-release (SAR) endolysin, a homolog of the T5 A1 protein required for DNA transfer, and a dual-start holin/antiholin pair.

Structure, dynamics and interactions of large SRP variants

Co-translational protein targeting to membranes relies on the signal recognition particle (SRP) system consisting of a cytosolic ribonucleoprotein complex and its membrane-associated receptor. SRP recognizes N-terminal cleavable signals or signal anchor sequences, retards translation, and delivers ribosome-nascent chain complexes (RNCs) to vacant translocation channels in the target membrane. While our mechanistic understanding is well advanced for the small bacterial systems it lags behind for the large bacterial, archaeal and eukaryotic SRP variants including an Alu and an S domain. Here we describe recent advances on structural and functional insights in domain architecture, particle dynamics and interplay with RNCs and translocon and GTP-dependent regulation of co-translational protein targeting stimulated by SRP RNA.

Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex

The Sec61 translocon provides an unexpectedly flexible and dynamic environment within which transmembrane regions of nascent polypeptides can be completely reoriented during the biosynthesis of multiple-spanning membrane proteins.

Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10−8 M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.