The mammalian caveolin gene family consists of caveolins-1, -2, and -3. The expression of caveolin-3 is musclespecific. In contrast, caveolins-1 and -2 are co-expressed, and they form a hetero-oligomeric complex in many cell types, with particularly high levels in adipocytes, endothelial cells, and fibroblasts. These caveolin hetero-oligomers are thought to represent the functional assembly units that drive caveolae formation in vivo. Here, we investigate the mechanism by which caveolins-1 and -2 form hetero-oligomers. We reconstituted this reciprocal interaction in vivo and in vitro using a variety of complementary approaches, including the generation of glutathione S-transferase fusion proteins and synthetic peptides. Taken together, our results indicate that the membranespanning domains of both caveolins-1 and -2 play a critical role in mediating their ability to interact with each other. This is the first demonstration that these unusual membrane-spanning regions found in the caveolin family play a specific role in protein-protein interactions.

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S⅐mRNA⅐ eIF3⅐Met-tRNA f ⅐eIF2⅐GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32 P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the ␤ subunit of initiation factor eIF2. No other initiation factors including the ␥ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the ␤ subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2␤ deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2␤. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNA f ⅐eIF2⅐GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the ␤ subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.Initiation of translation in eukaryotic cells occurs by a sequence of partial reactions requiring the participation of a large number of specific proteins called eukaryotic (translation) initiation factors (eIFs).1 An obligatory intermediate step in this overall initiation reaction is the binding of the initiator methionyl-tRNA (Met-tRNA f ) as Met-tRNA f ⅐eIF2⅐GTP ternary complex to a 40 S ribosomal subunit, followed by positioning of the 40 S preinitiation complex (40 S⅐Met-tRNA f ⅐eIF2⅐GTP) at the initiation AUG codon of the mRNA to form the 40 S initiation complex (40 S⅐mRNA⅐Met-tRNA f ⅐eIF2⅐GTP). A 60 S subunit then joins the 40 S initiation complex to form the 80 S initiation complex (80 S⅐mRNA⅐Met-tRNA f ) that is active in peptidyl transfer (for a review, see Refs. 1-3). The subunit joining reaction specifically requires the participation of eIF5, an initiation factor that we have purified and characterized from mammalian cells (4 -6) and the yeast Saccharomyces cerevisiae (7). Detailed characterization of the eIF5-catalyzed reaction has shown that eIF5 first interacts with the 40 S initiation complex in the absence of 60 S ribosomal subunits to promote the hydrolysis of ribosome-bound GTP (8). Hydrolysis of GTP causes the release of eIF2 and guanine nucleotide (as an eIF2⅐GDP complex) from the 40 S subunit, an event that is essential for the subsequent joining of the 60 S ribosomal subunit to the 40 S complex (40 S⅐mRNA⅐Met-tRNA f ) to form the 80 S initiation complex that is active in subsequent peptidyl transfer reaction (8 -11).The mammalian cDNA (rat and human) and the S. cerevisiae gene encoding eIF5 of calculated M r ϭ 48,926 and 45,346, respectively, have been cloned and expressed in Escherichia coli (12)(13)(14)(15)). An interesting feature of the derived amino acid sequences of mammal...

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