Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling

Abstract

Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mistargeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3␤ and ap-4␤ knock-out mutants. In ap-3␤ mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4␤ mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3␤ and ap-4␤ compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking. Molecular & Cellular

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine‐based motif

“…We could demonstrate that GFP‐fusions of the tonoplastic proteins MOT2, as well as NRAMP3 and NRAMP4, are partially missorted to the PM and to intracellular compartments in ap4ß‐1 and ap4μ mutants, while GFP‐INT1 is not, and that a WT‐like distribution is re‐established in stable transformants of ap4μ expressing AP4μ Pro :AP4μ‐GUS. Partial missorting of NRAMP3 and NRAMP4 in ap4 mutants might contribute to reduced chlorophyll contents and to depletion of cytochrome p450 proteins in ap4 mutants reported earlier, either by impairing the release of iron from the vacuole, or by disturbing iron distribution between cells or tissues via mislocalized NRAMP3, and particularly NRAMP4. This could explain why stable overexpression of NRAMP4‐GFP in ap4μ plants did not complement chlorophyll concentration of seedlings grown on iron‐deficient medium (Figure S1 in Appendix S1).…”

“…plants to test for potential additive effects, and established stably transformed ap4μ mutants expressing the genomic sequence of AP4μ fused to GUS or GFP under control of the AP4μ ‐promoter ( ap4μ/AP4μ Pro :AP4μ‐GFP and ap4μ/AP4μ Pro :AP4μ‐GUS ) as complementation lines. Measurement of primary root lengths revealed that, identical to ap4ß‐1 mutants, ap4ß‐2 and ap4μ mutants also developed significantly shorter roots compared to the WT (Figure A). Moreover, all single and double k.o.…”

“…(ecotype Col‐0) was used as control for all experiments. The T‐DNA insertion lines ap4ß‐1 (SAIL_781_H01, identical with gfs4‐4 43 and with ap‐4ß ), ap4ß‐2 (SAIL_796_A10, identical with gfs4‐3 ) and ap4μ (SALK_014326, equivalent to gfs5‐3 ) were obtained from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/). Single and double k.o.…”

“…To analyze the function of Arabidopsis AP4 in plant development, we examined mutant lines carrying a T-DNA insertion in the gene coding for the ß-subunit (ap4ß-1 44 and ap4ß-2 43 ) or the medium subunit (ap4μ 43 ) of AP4. We further created ap4ß-2 ap4μ double knock-out (k.o.)…”

“…Information on developmental defects of ap4 mutants, however, is still limited. The only morphological differences between Arabidopsis ap4 mutants and wild‐type (WT) described to date are general dwarfism and short roots, but the latter has been examined in only one mutant line lacking the ß‐adaptin of Arabidopsis AP4. More comprehensive analyses were thus not only fundamentally important, but will also contribute to the identification of further cargo proteins and sorting pathway(s) of the complex.…”