CytLEK1 Is a Regulator of Plasma Membrane Recycling through Its Interaction with SNAP-25

Abstract

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

Rab11-FIP2 regulates differentiable steps in transcytosis

“…Whereas we could not determine any discrete alteration of interactions with FIP2(R413G), it is also possible that the R413G mutation may lead to stabilization of higherorder trafficking complexes. Similar inhibition of plasma membrane through stabilization of complexes was recently suggested for overexpression of a combination of cytoplasmic Lek1 (cytLEK1) and synaptosomal-associated protein of 25 kDa (SNAP-25) (26). A number of studies including this work have suggested that trafficking through the recycling system may be regulated by multiple hand-offs between regulatory complexes defining discrete subdomains within the plasma membrane recycling system (14).…”

“…This mutant has been used by us and others to assess proteins utilizing the Rab11a recycling pathway (8,11,19,20). Similarly, a mutant myosin Vb lacking the motor domain, myosin Vb-tail, also acts as a dominant-negative mutant and has proven useful in the assessment of trafficking through the Rab11a trafficking pathway (4,26,32,33). However, we recently have reported that FIP2 also has a role in the establishment of cell polarity (7).…”

Acrosome formation-associated factor is involved in fertilization

“…Take together with the observed interaction of Afaf and SNAP25, we concluded that Afaf and SNAP25 might form a negative complex and resulted in an inability of the cells to uptake Tf properly. Interestingly, the similar finding was also observed in the complex of cytLEK and SNAP25 (19).…”

“…To address this question, HeLa cells were then cotransfected with both Afaf-myc and a GFP-SNAP25 fusion construct. As seen in Figure 4A, GFP-SNAP25 expressing cells showed a cytoplasmic distribution with high levels of fluorescence observed in the perinuclear region and at the cell periphery, which was similar with the expression pattern of SNAP25 in other cell types (17,19). The cotransfected cells demonstrated an extensive overlap of SNAP25 with Afaf, as seen in the merged image, but Afaf showed a specific distribution into the cell membrane.…”

“…Endocytic trafficking has also been reported to contribute to acrosome formation (18). As a second role of SNAP25 (17), Cytoplasmic Lek1 (cytLEK1) was identified as a link between recycling vesicles and the microtubule network through its association with SNAP-25 and Nde1 (19).…”