Although exocytosis is critical for the proper trafficking of materials to the plasma membrane, relatively little is known about the mechanistic details of post-Golgi trafficking in plants. Here, we demonstrate that the DENN (Differentially Expressed in Normal and Neoplastic cells) domain protein STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) and SCD2 form a previously unknown protein complex, the SCD complex, that functionally interacts with subunits of the exocyst complex and the RabE1 family of GTPases in Consistent with a role in post-Golgi trafficking, and mutants display defects in exocytosis and recycling of PIN2-GFP. Perturbation of exocytosis using the small molecule Endosidin2 results in growth inhibition and PIN2-GFP trafficking defects in and mutants. In addition to the exocyst, the SCD complex binds in a nucleotide state-specific manner with Sec4p/Rab8-related RabE1 GTPases and overexpression of wild-type RabE1 rescues temperature-sensitive mutants. Furthermore, SCD1 colocalizes with the exocyst subunit, SEC15B, and RabE1 at the cell plate and in distinct punctae at or near the plasma membrane. Our findings reveal a mechanism for plant exocytosis, through the identification and characterization of a protein interaction network that includes the SCD complex, RabE1, and the exocyst.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

The plant UBX-domain containing protein 1, PUX1, functions as a negative regulator of gibberellin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a “GA-overdose” phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor, GA-INSENSITIVE DWARF-1, GID1. This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway. Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric AAA+ ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation for proteasomal degradation, cell division and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays. Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.