Insertion Mechanism in E. Coli of Single-Span Membrane Proteins with Far-Downstream Transmembrane Segments

Abstract

Single-span membrane proteins (SSMPs) are the most abundant membrane proteins in virtually all organisms. SSMPs with N-terminus outside the cell are referred to as Type I MPs, while those with the N-terminus inside are referred to as Type II MPs. It is generally assumed that insertion occurs co-translationally via the signal recognition particle (SRP) pathway. The positive-inside rule determines TypeI/II MPs. The TM segment of Type I MPs is generally preceded by a cleavable signal sequence to facilitate the Nout-Cin topology. The TM segment of both types of proteins generally occurs early in the amino acid sequence, allowing recognition by SRP as it emerges from the ribosome. Secreted periplasmic proteins have a signal sequence, but are secreted post-translationally through the translocon by the SecA translocase.We are studying an unusual class of Type II MPs that lack identifiable signal sequences and whose TM segments can occur hundreds of residues downstream from the N-terminus. One such protein is RodZ, which is a cytoskeletal protein involved in maintaining the rod shape of E. coli. What pathway does this protein use? Several prediction programs and our experimental studies of RodZ with deleted TM segment (RodZ-ΔTM) confirm the absence of a native targeting signal. Proteinase K treatment of spheroplasts reveal that RodZ has Nin-Cout (Type II) topology, consistent with previous studies. Classic N-terminal cleavable signal sequences, such as DsbA or MalE, do not affect the topology of RodZ. We have determined that RodZ insertion is strongly SecA dependent, because RodZ is not incorporated into the membrane under SecA-depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. This result is important, because it is now possible to study the energetics of TM helix insertion by SecA.