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Figure 3


Fig. 3. The dileucine-like motif in i3 functions as a signal sufficient for lysosomal sorting in HeLa cells. (A) Structure, sequence and cross-species conservation of the LAMP/i3FY construct, carrying the 19 N-terminal amino acids of i3, and of its deletion and mutagenesis variants. N-term, N-terminus of rat LAMP1; TM, transmembrane domain of rat LAMP1; these were fused with the cytosolic loop of OA1. (B) Immunofluorescence analysis of HeLa cells transiently transfected with expression vectors for LAMP/i3 chimeras and immunodecorated with anti-rat LAMP1 antibodies. Cells were counterstained with the fluid-phase marker Lucifer Yellow at 1 hour chase prior to fixation to label the lysosomes (L.Y.), or were cotransfected with an expression vector for farnesylated GFP to label the plasma membrane (EGFP-F). In the merge, areas of colocalization can be observed in yellow/orange. LAMP/i3FG behaves as LAMP/i3FY LL->AA (not shown). LAMP/i3{Delta}FY is a LAMP/OA1 chimera carrying the 18 C-terminal amino acids of i3. Bar, 10 µm.





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