Short N-terminal sequences package proteins into bacterial microcompartments

Abstract

Hundreds of bacterial species produce proteinaceous microcompartments (MCPs) that act as simple organelles by confining the enzymes of metabolic pathways that have toxic or volatile intermediates. A fundamental unanswered question about bacterial MCPs is how enzymes are packaged within the protein shell that forms their outer surface. Here, we report that a short N-terminal peptide is necessary and sufficient for packaging enzymes into the lumen of an MCP involved in B 12 -dependent 1,2-propanediol utilization (Pdu MCP). Deletion of 10 or 14 amino acids from the N terminus of the propionaldehyde dehydrogenase (PduP) enzyme, which is normally found within the Pdu MCP, substantially impaired packaging, with minimal effects on its enzymatic activity. Fusion of the 18 N-terminal amino acids from PduP to GFP, GST, or maltose-binding protein resulted in their encapsulation within MCPs. Bioinformatic analyses revealed N-terminal extensions in two additional Pdu proteins and three proteins from two unrelated MCPs, suggesting that N-terminal peptides may be used to package proteins into diverse MCPs. The potential uses of MCP assembly principles in nature and in biotechnology are discussed.

Phylogeny and physiology of candidate phylum ‘Atribacteria’ (OP9/JS1) inferred from cultivation-independent genomics

“…Although such aldolases and sugar isomerases are not typically associated with BMC loci in other organisms, comparison of the predicted products of these genes with close homologues revealed an N-terminal 30-40 amino-acid sequence unique to the BMC-associated 'Atribacteria' proteins (Supplementary Figure S4). This suggests that they may be targeted to the BMC lumen, as several BMC-associated genes from other bacteria have been shown to have N-terminal extensions of similar length for facilitating BMC lumen localization, although prediction of specific residues responsible for targeting is not straightforward (Fan et al, 2010). A putative transcriptional regulator flanking the BMC cluster in the Sakinaw SAG co-assembly (Figure 3) was also identified as a conserved 'Atribacteria' gene (Supplementary Table S4).…”

Addressing microbial organelles: A short peptide directs enzymes to the interior

“…Such contacts would explain the regular arrangement of carboxysomal RubisCO molecules in concentrical layers and the fact that the interior of the great majority of carboxysomes appears to be solidly filled with proteins (17). Specific interactions between BMC components that are not in direct contact with the BMC shell could also explain why traces of mutant PduP lacking the N-terminal peptide were found to be present in isolated BMCs by Fan et al (5).…”

“…The specificity of the packaging signal was tested with a construct featuring the first 18 residues of the N-terminal extension. By controlling the levels of the fusion protein in a WT S. enterica background, Fan et al (5) show that increasing amounts of GFP are packaged into the Pdu BMC in competition with endogenous PduP as expression levels of the fusion protein increase.…”

“…Fan et al (5) suggest that the Nterminal sequences of PduP proteins and similar extensions of five other BMC proteins may contribute to a general mechanism for BMC sequestration that depends on specific interactions of the different packaging signals with individual…”

“…The report by Fan et al (5) leads the way for searches of additional packaging signals in other types of BMCs and for the development of these bacterial organelles toward biotechnological applications.…”