FIG. 1.

(A) Map of the IAP-specific sequence in plasmids pTM1-MIA2, pTM1-MIA4, and pTM1-MIA5. MIA2 corresponds to the wild-type sequence of the IAP MIA14 (38). In MIA4, the first 10 codons of src were substituted for the first 28 codons of the gag gene, and in MIA5 the first 28 codons of gag were deleted. Nucleotide numbering is according to the published sequence (38). Proteins derived from different reading frames are depicted in different lanes. (B) Analysis of in vitro-translated and membrane-bound IAP polyproteins. Coupled transcription-translation reactions were programmed with pTM1-MIA2, pTM1-MIA4, or pTM1-MIA5 in the absence or presence of microsomal membranes as indicated and subsequently centrifuged for 15 min at 12,000 × g. Supernatants (S) and resuspended pellets (P) were applied in equivalent amounts and analyzed by SDS-PAGE. Translation products were labeled with [³⁵S]methionine and detected by autoradiography. The positions of the Gag, Gag-PR, and Gag-PR-Pol polyproteins are indicated on the left. (C) Analysis of membrane-bound IAP polyproteins by sucrose flotation. Coupled transcription-translation reactions were programmed with pTM1-MIA2 or pTM1-MIA5 in the absence or presence of microsomal membranes as indicated. After translation, the samples were adjusted to 85% (wt/vol) sucrose and overlaid with 65 and 10% sucrose. The step gradient was centrifuged at 43,000 rpm in a Beckman Optima TLX ultracentrifuge for 18 h at 4°C. Fractions were collected from top (fraction 1) to bottom (fraction 8), and proteins were resolved by SDS-PAGE.