MCD BIO 165A Lecture 5: Li quiz

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Molecular, Cell, and Developmental Biology

1. a) In testing if dileucine motifs are required for targeting specificity the authors truncated the Ser1 protein so that it either lost the dileucine motif or truncated another part of the protein so that the dileucine motif stayed intact (2a). In the truncated version that lost the motif, Ser1 became diffuse and lost specificity but with the motif still intact the protein maintained axonal specifity. Furthermore, as shown in both 2a and 2b, the authors mutated the dileucine motif of Ser1 directly replacing the LI with AA, a neutral residue. They then coexpressed Ser1 GFP with cystolic td Tomato and saw that Ser1 lost its preferencespecificity in the cell. This suggests that the dileucine motif is required for sorting the proteins in a polarized manner. b) To test that the dileucine motif was sufficient for Ser1 localization, they fused Ser1 cytoplasmic domain to mCD8, a generic nonpolarized transmembrane domain. This localized in the axon, mutated this sequence by adding two alanines in place of the Ll and this caused diffuse localization. Next, they added the cytoplasmic domain of the dendritic protein GLR1, which was found sufficient to lead to dendritic localization. Lastly, they completely replaced the Ser1 motif with a dendritic motif while keeping the rest of the Ser1C sequence intact. DNSLL and DLHELL (the dendritic motifs) were able to cause Ser1 to localize to the dendrites. Therefore, it seems that the dileucine motif is specifically directed to localize the proteins and is sufficient. 2. The purpose of the experiment was to answer the question: does AP3 binding efficiency correlate with axonal localization? And, it is also meant to answer the broader question: do dileucine motifs have selective interactions with AP complexes? In the experiment, mutating a nonleucine amino acid in the dileucine variable region makes differing dileucine motifs, which have variable binding to AP3. After mutating the threonine in the variable region, they FLAG tagged AP3 pulled down it with Ser1GTP mutants. Additionally, they quantified the relative binding efficiencies and polarity indexes of the Ser1 variants. They found that AP3 binding affinity correlates with axonal location: low affinity directs towards the dendrites and a diffuse distribution of the protein while high affinity directs it to the axons. 3. In Figure 5F, AP3 localized to a perinuclear domain in the Golgi, which is adjacent but not overlapping to the AP1 domain, so AP3 and AP1 sort the axonal proteins are sorted into different vesicles from the Golgi. According to Figure 5I, the kymographs of axonal (Rab3) and dendritic (GLR1) proteins show that GLR1 moves slower than Rab3 vesicles and that axonal and dendritic proteins are separated into different postgolgi carriers. The overlap between the two kymographs show they are being sorted into different vesicles at different speeds and in different positions, which suggests that axonal and dendritic vesicles for these proteins do not overlap. However, if one compares this information to the data from Figures 6 A and F, it is show that when Ap3 is mutated some of the axonal and dendritic proteins localize to the same vesicles as show by the yellow in the kymograph. Therefore, in mutated Ap3 there is defective separation of axonal and dendritic proteins in the soma. Figures 6D and 6F show that in Ap3 mutants, the average speed of Rab3 is reduced to that of GLR1 but the speed of GLR1 does not change. This suggests that Ap3 is required to select cargos into the fastmoving axonal vesicles. In the case of mutant Ap3, Ap1 does bind axonal proteins although with lower affinity. Also, Figure 6G shows that in reference to the Ap3 mutant, Ap3 and Ap1 double
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