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. 2002 Aug 19;158(4):659–668. doi: 10.1083/jcb.200204081

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Copyright © 2002, The Rockefeller University Press

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Figure 4. — Only Rab32 and RabRP-1 contain determinants for PKA anchoring. (A) The sequences of Rab32 and nine other Rab proteins are aligned within the α5 helix region. The first and last residues of each sequence and the name of each Rab protein are indicated. Boxed and shaded regions depict sequence identity and similarity. Star indicates the conserved phenylalanine in most Rab proteins. (B) A solid-phase peptide array of these Rab sequences was screened for RII binding by the overlay assay. Binding of [³²P]RIIα was detected by autoradiography. (C) Quantitation of RII binding was assessed by densitometry. Arbitrary units were normalized to one, indicating the highest level of RII binding. The data presented are an amalgamation of two independent experiments. (D) Yeast two-hybrid analysis was used to determine if RII interacts with selected, full-length Rab family members. Two-hybrid crosses of RIIα 1–45 fragment with full-length cDNAs for Rab32, Rab32L188P, Rab5, Rab6, or Rab7 fused to the LexA DNA binding domain. Interactions were assayed by growth on minimal media plates in the absence of histidine (left). As a toxicity control, all cotransformed yeast strains were able to grow in the presence of histidine (right). (E) HEK-293 cell extracts expressing Flag-tagged Rab32 and mutants (indicated above each lane) were separated by SDS-PAGE on 4–15% gels and electrotransferred to nitrocellulose. Binding of [³²P]RIIα was assessed by the overlay assay. (Top) RII binding was detected by autoradiography. Equal loading of recombinant Rab32 proteins was confirmed by Western blot using antiFlag antibodies. (F) Sequence alignment of Rab32 and its most closely related family members. The first and last residues of each sequence and the name of each Rab protein are indicated. Boxed and shaded regions depict sequence similarity and identity. Star indicates the position of alanine 185 in the Rab32 sequence. (G) A solid-phase peptide array of these closely related Rab sequences was screened for RII binding by the overlay assay. Binding of [³²P]RIIα was detected by autoradiography.