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Transport properties of canonical PIN proteins and the role of the loop in auxin transport

ABSTRACT: Auxin is a pivotal plant hormone that controls virtually all aspects of plant growth and development. Auxin is transported in plants in a polar fashion and PIN-FORMED (PIN) transporters are the key players in this process. Canonical PINs possess a long cytosolic loop that is subject to regulation by AGC1 and AGC3 kinases. To characterize the transporters, they are expressed in Xenopus laevis oocytes alone or with the different activating kinases. Since transport rates always depend on the amount of active transporter in the membrane it is paramount to ensure that the levels of transporter, kinase and degree of phosphorylation are similar to be able to compare transport rates directly. To this end we expressed PIN1, PIN2 and PIN3 either alone or with D6PK (an AGC1 kinase) or PID (an AGC3 kinase) in oocytes. The protein levels and the degree of phosphorylation of the loop were established and found to be comparable between PINs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Oocyte

SUBMITTER: Julia Mergner

LAB HEAD: Bernhard Kuster

PROVIDER: PXD044850 | Pride | 2024-08-23

REPOSITORIES: Pride

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			Action	DRS
	03141_BA10_P035673_S00_A00_R1.raw	Raw
	03141_BA11_P035681_S00_A00_R1.raw	Raw
	03141_BA12_P035689_S00_A00_R1.raw	Raw
	03141_BA3_P035634_S00_A00_R1.raw	Raw
	03141_BA4_P335638_S00_A00_R2.raw	Raw

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Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are co-infected with Epstein-Barr Virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen (EBNA)-2 to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV+/EBV+ PEL cells, but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA2 growth deficiency in an autocrine/paracrine manner. Complementaton of EBNA2-deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV+/EBV+ PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA2 and Rta induce distinct alterations in the cellular proteomes that contribute to growth of infected cells. EREB2-5 cells were transfected and grown in the presence or absence of β-estradiol, as described. Seven days post-transfection, protein extracts were prepared, and 200 ugs. of each were analyzed using the RayBio Human Apoptosis Antibody Array Kit (RayBiotech) as per manufacturers suggestions. The membranes were exposed to autoradiography film for different times to detect the chemiluminescent signals. Images with signals in linear range were quantitated using the program ImageJ [59]. For each membrane, signals from the negative control spots were averaged, and then subtracted from each of the other spots. A signal was considered valid if its value exceeded both its average local background, and the average of all valid negative control values. Valid signals were normalized using the positive control spots (for cellular BID protein). Fold change in signals for each spot were quantitated by dividing by the valid signals for each corresponding spot on the minus β-estradiol membrane. Average fold change, and standard deviation, were calculated for each protein.

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Transport properties of canonical PIN proteins and the role of the loop in auxin transport

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