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Fig. 4. LUMA topology. (A) Model for LUMA topology with cytoplasmic or nucleoplasmic termini and a large lumenal hydrophilic domain. (B) Scheme of LUMA constructs: full-length LUMA 1-400 bears a C-terminal V5-His6-tag, the other constructs contain a Myc-His6-tag. N-terminal truncation mutants starting with LUMA residue 53 received a N-terminal signal peptide (SP; grey box) for ER export. Anti-LUMA antibodies recognize the hydrophilic domain of LUMA (indicated by the bar above the scheme for LUMA 1-400), which is replaced by a part of β-galactosidase in the LUMAgal mutant (gal; grey box). Black boxes indicate TMDs. (C) Full-length LUMA and LUMA 1-309 were ³⁵S-labeled by coupled in vitro transcription and translation in the presence of microsomes, and then digested with proteinase K. Remaining fragments were separated by SDS-PAGE and visualized by autoradiography. a, full-length LUMA 1-400 V5/His; b, 42.5-kDa fragment of LUMA protected from proteolysis by the membrane; c, LUMA 1-309 and fragment; d, by-product of LUMA 1-309 translation; e and f, fragments generated from LUMA not inserted into microsomal membranes.

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