Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.
Bottom Line: Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity.Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ~70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ~100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins.Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.
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Mentions: Because the hydrophobic NA TMDs (ΔGapp < 0 kcal/mol) with short, 36-aa C-tails properly localized to the PM (Figures 2 and 3), we also examined the hydrophobic NA TMD orientation with increasing C-tail lengths. At steady-state, these constructs were already ∼65% glycosylated, with a C-tail of 36 aa, and their glycosylation percentage increased further once the C-tail length was >61 aa (Figure 5A, top, immunoblots, and B). The analysis of the nascent chains after a 15-min pulse showed that the percentage of glycosylated species increased with the C-tail length beginning from 36 aa (Figure 5A, bottom, audioradiographs, and B). These results demonstrate that hydrophobic NA TMDs benefit from, but do not require, the cotranslational membrane insertion process (ribosomal attachment and/or synthesis) for their inversion, which explains their PM localization with short C-tails.