Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone

26-Norbrassinolide, identified as a metabolite of brassinolide in cultured cells of the liverwort, Marchantia polymorpha, as well as 26-norcastasterone and 26-nor-6-deoxocastasterone were synthesized. Synthesis of these new brassinosteroids was conducted by employing the orthoester Claisen rearrangement and asymmetric dihydroxylation as key reactions. The modified rice lamina inclination test indicated that these three 26-norbrassinosteroids were less active than their corresponding C28 brassinosteroids. Growth-promoting activities were also examined by using the brassinosteroid-deficient, dwarf mutant lkb of garden pea (Pisum sativum L.). In this assay, 26-norbrassinolide was as effective as brassinolide and 26-norcastasterone was more effective than castasterone although 26-nor-6-deoxocastasterone was much less effective than 6-deoxocastasterone. Therefore, removal of C-26 of brassinosteroids does not necessarily reduce the biological activity. The role of C-26 removal in Marchantia cells remains unclear.     

Vps26A and Vps26B subunits define distinct retromer complexes

The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.