Figure 5

Functional analysis of a nuclear export signal in 16.4.1. (A) Depicted is the sequence (nucleotides and predicted amino acids) of the 16.4.1 protein investigated here. The sequence encoding amino acids 1–8 are derived from the fetal heart cDNA W67699. The region between amino acid residue 74 and 133 showing CRM1-dependent nuclear export activity (Fig. 4) is underscored. The amino acid sequence between residues 86 and 105 (shaded in grey) contains several Leucine and Isoleucine residues representing a candidate nuclear export signal. (B) Functional characterisation of the Leucine-Isoleucine rich sequence of 16.4.1. Comparison of the translocation activities of 16.4.1 region 86–105 and the Rev-NES in a microinjection-based transport assay [51]. Transport substrates were generated by conjugating peptides containing region 86–105 of 16.4.1 or region 73–84 of Rev with bovine serum albumin (BSA) labeled with a red fluorescent dye. Transport substrates were coinjected into the nucleus with an injection control consisting of unconjugated BSA labeled with a different fluorescent dye (e.g. green). The proportion of each fluorescent label in the nucleus of the injected cell was determined and the ratio of fluorescence of the transport substrate to fluorescence of the injection control calculated. This ratio represents the relative translocation activity of the transport substrate and is indicated in the graph. Nuclear export activity yields ratios <1 as demonstrated for the Rev NES. Transport substrates containing amino acid region 86–105 of 16.4.1 also yielded a relative translocation activity <1, indicating that this region of 16.4.1 can function as a nuclear export signal.