Background: Traffic of the amyloid precursor protein (APP) determines its processing to neurotoxic A␤ peptides. Results: Mint3 is recruited to the Golgi by APP and is required for export to a LAMP1 ϩ compartment.

Background: Cargo proteins recruit Arf-dependent adaptors for packaging. Results: Cargo presence at the Golgi/endosomes leads to specific adaptor recruitment. Conclusion: Arf activation is more closely coupled to cargo than previously appreciated, and we interpret the specificity in adaptor recruitment as evidence of the lack of freely diffusible activated Arfs. Significance: Cargos play a role in the activation of Arfs and recruitment of specific adaptors.

The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.

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