The Syt VII membrane proximal cysteines C35, C38, and C41 are required for complete lysosomal localization. NRK cells transduced with adenovirus expressing Lamp1-RFP and either Syt VII–YFP, Syt VII (C35S)–YFP, Syt VII (C38S)–YFP, Syt VII (C41S)–YFP, Syt VII (C35/38S)–YFP, Syt VII (C35/41S)–YFP, Syt VII (C38/41S)–YFP, or Syt VII (C/S)–YFP (green) were imaged by live confocal microscopy. Images represent maximum-intensity projections of 16 Z sections. Cells expressing Syt VII single-cysteine mutants display partial Syt VII localization to lysosomes and Golgi, whereas cells expressing Syt VII double- and triple-cysteine mutants display complete Golgi retention of Syt VII. Bar, 6 µm.