Andreas Weise

SUT2, a Putative Sucrose Sensor in Sieve Elements

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter–like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

A New Subfamily of Sucrose Transporters, SUT4, with Low Affinity/High Capacity Localized in Enucleate Sieve Elements of Plants

A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The Km for sucrose uptake by AtSUT4 of 11.6 ± 0.6 mM was ∼10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

Alternative splicing of Tcf7l2 transcripts generates protein variants with differential promoter-binding and transcriptional activation properties at Wnt/β-catenin targets

Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of β-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/β-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/β-catenin signalling.

Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity

AtSUT2 was found to be a low‐affinity sucrose transporter (K M=11.7 mM at pH 4). Chimeric proteins between AtSUT2 and the high‐affinity StSUT1 were constructed in which the extended N‐terminus and central loop of AtSUT2 were exchanged with those domains of StSUT1 and vice versa. Chimeras containing the N‐terminus of AtSUT2 showed significantly lower affinity for sucrose compared to chimeras containing the N‐terminus of StSUT1. The results indicate a significant function of the N‐terminus but not the central cytoplasmic loop in determining substrate affinity. Expression of AtSUT2 in major veins of source leaves and in flowers is compatible with a role as a second low‐affinity sucrose transporter or as a sucrose sensor.

High trefoil factor 1 (TFF1) expression in human retinoblastoma cells correlates with low growth kinetics, increased cyclin-dependent kinase (CDK) inhibitor levels and a selective down-regulation of CDK6

Trefoil factor family (TFFs) peptides facilitate epithelial restitution, but also effect cell proliferation and apoptosis of normal and various cancer cell lines. In a recent study by our group, TFF2 expression was demonstrated in the murine retina, where it exhibits pro-proliferative and pro-apoptotic effects. In the present study, we investigated the expression and function of TFF peptides in eight human retinoblastoma cell lines. TFF1 was the only TFF peptide expressed at detectable levels in immunoblots of retinoblastoma cells. TFF1 expression levels were highly variable in different retinoblastoma cell lines and negatively correlated with cell growth curves. Recombinant human TFF1 had a negative effect on cell viability and caused a reduction in cell proliferation. Retinoblastoma cell lines with high TFF1 expression levels exhibited a selective down-regulation of cyclin-dependent kinase (CDK) 6, whereas CDK4 and CDK2 seem to be unaffected by TFF1 expression. In immunocytochemical studies, we observed a nuclear co-localization of TFF1 and CDK2 in Cajal bodies (CBs). In high TFF1 expressing human retinoblastoma cell lines CBs were smaller and higher in number compared to retinoblastoma lines with low TFF1 expression, indicating differences in cell cycle status between the different retinoblastoma cell lines. Our data further support the notion for a potential tumor suppressor function of TFF1. The nuclear localization of TFF1 in CBs—considered to play a role in cell cycle progression, potentially acting as a platform for CDK-cyclin function—offers a new impetus in the ongoing search for potential TFF1 interacting proteins.