Abstract
The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.
Highlights
Prokaryotic cytoplasmic membrane proteins represent 20–30% of the proteome (Wallin and von Heijne, 1998; Krogh et al, 2001) and they fulfil a wide variety of critical functions in the cell including respiration, photosynthesis, and ion transport, allowing this membrane to act as a tightly controlled barrier between the cytoplasm and the extracellular environment
To assess the mechanism of transmembrane domains (TMDs) insertion we used constructs where the cofactor-containing FeS domain was genetically removed from Sco2149 and replaced with the mature region of two different reporter proteins – that of the E. coli Tat substrate AmiA (Ize et al, 2003) to report on interaction of Sco2149 with the Tat pathway, or of the Sec substrate b-lactamase (Bla, which is compatible for export with either the Sec or Tat pathways depending on the nature of the targeting sequence [Stanley et al, 2002]) (Figure 1A, Figure 1—figure supplement 1)
In a previous study we identified the actinobacterial Rieske FeS protein as the first protein known to be targeted to the plasma membrane by the dual action of the Sec and Tat translocases
Summary
Prokaryotic cytoplasmic membrane proteins represent 20–30% of the proteome (Wallin and von Heijne, 1998; Krogh et al, 2001) and they fulfil a wide variety of critical functions in the cell including respiration, photosynthesis, and ion transport, allowing this membrane to act as a tightly controlled barrier between the cytoplasm and the extracellular environment. The insertion of transmembrane domains of polytopic proteins occurs co-translationally following targeting of the translating ribosome to the Sec machinery through the action of signal recognition particle (SRP) (Ulbrandt et al, 1997). YidC can act independently of the Sec system to integrate small (usually mono- or bitopic) membrane proteins directly into the bilayer (Dalbey et al, 2014; Samuelson et al, 2000). The final topology adopted by a polytopic membrane protein depends upon a number of intrinsic and extrinsic factors including the hydrophobicity of membrane-spanning regions, the number and location of positively-charged amino acids and the composition of the lipid bilayer (White and von Heijne, 2008a; Cymer et al, 2015; Bogdanov et al, 2014)
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