Table 1. List of constructs used.

figure	name	gene	source	No. TMS	insertion machinery	modifications	vector
Fig.2	β-lactamase	β-lactamase	E. coli	None (secreted)	translocon	Strep-tag sequence fused to the C-terminus of β-lactamase	pTD1-strep
	KvAP	K+ channel	Aeropyrum pernix	6		prolactin containing g-loop fused to cytosolic C-terminus of KvAP. G-loop at extracellular loop between S3 and S4	pTD1
	pro-TNF	pro-tumor necrosis factor	human	1		N-glycosylation site at the extracellular C-terminal region	pTD1
	Syb2	synaptobrevin II	human	1	GET	Drosophila-based GVPYVSSSDSGSGGGNKNITQAPPH sequence containing engineered glycosylation site at C terminal region after transmembrane of Syb2	pTD1
Fig.3	AtKC1	K+ channel	Arabidopsis thaliana	6	translocon	Strep-tag sequence fused to the C-terminus of each protein	pTD1-strep
	KAT1						pTD1-strep
	KAT2						pTD1-strep
	GORK						pTD1-strep
	AKT1						pTD1-strep
	NhaA	Na+/H+ antiporter	E. coli	12		Strep-tag sequence fused to the C-terminus of NhaA.	pTD1-strep
	AspT	aspartate: alanine antiporter	Tetragenococcus halophilus	10		6× His tag sequence at the C-terminus	pTD1
	MsbA	ABC transporter	E. coli	6		6× His tag sequence at the N-terminus	pTD1
	Sec61β	Sec61β	human	1	GET	Drosophila-based GVPYVSSSDSGSGGGNKNITQAPPH sequence containing engineered glycosylation site at C terminal region after transmembrane of Sec61β	pTD1
Fig. 4	AspT-His	AspT-His	Tetragenococcus halophilus	10	translocon	6× His tag at a cytoplasmic large loop between the 5th and the 6th transmembrane domain	pTD1
Fig. 5A, 5B	NhaA	Na+/H+ antiporter	E. coli	12	translocon	8× His tag sequence at the C-terminus	pTD1
Fig. 5C	NhaA	Na+/H+ antiporter	E. coli	12	translocon	Addition of Gly-Gly, non-natural amino acid immediately after V388 in the NhaA construct as shown in Fig. 3.	pTD1-strep
Fig. 6	KvAP	K+ channel	Aeropyrum pernix	6	translocon	See Fig. 2.	pTD1
	pro-TNF	pro-tumor necrosis factor	human	1		See Fig. 2.	pTD1
	Syb2	synaptobrevin 2	human	1	GET	See Fig. 2.	pTD1