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Recycling of Solanum Sucrose Transporters Expressed in Yeast, Tobacco, and in Mature Phloem Sieve Elements

Molecular Plant 2010年第6期3卷 1064-1074页

作者： Johannes Liesche Hong-Xia He Bernhard Grimm Alexander Schulz christina kuhn Humboldt University Institute of Biology Plant Physiology Philippstraβe 13 Building 12 10115 Berlin Germany Plant Physiology and Anatomy Laboratory Department of Plant Biology Faculty of Life Sciences Copenhagen University Thorvaldsensvej 40 DK-1871 Frederiksberg C Denmark

The plant sucrose transporter SUT1 （from Solanum tuberosum, S. lycopersicum, or Zea mays） exhibits redoxdependent dimerization and targeting if heterologously expressed in S. cerevisiae （Krtigel et al., 2008）. It was also shown that SUT1 is present in motile vesicles when expressed in tobacco cells and that its targeting to the plasma membrane is reversible. StSUT1 is internalized in the presence of brefeldin A （BFA） in yeast, plant cells, and in mature sieve elements as confirmed by immunolocalization. These results were confirmed here and the dynamics of intracellular SUT1 localization were further elucidated. Inhibitor studies revealed that vesicle movement of SUT1 is actin-dependent. BFA-mediated effects might indicate that anterograde vesicle movement is possible even in mature sieve elements, and could involve components of the cytoskeleton that were previously thought to be absent in SEs. Our results are in contradiction to this old dogma of plant physiology and the potential of mature sieve elements should therefore be re-evaluated. In addition, SUT1 internalization was found to be dependent on the plasma membrane lipid composition. SUT1 belongs to the detergent-resistant membrane （DRM） fraction in planta and is targeted to membrane raft-like microdomains when expressed in yeast （Kr（igel et al,, 2008）, Here, SUT1-GFP expression in different yeast mutants, which were unable to perform en- docytosis and/or raft formation, revealed a strong link between SUT1 raft localization, the sterol composition and mem- brane potential of the yeast plasma membrane, and the capacity of the SUT1 protein to be internalized by endocytosis. The results provide new insight into the regulation of sucrose transport and the mechanism of endocytosis in plant cells.

关键词： Transporters membrane proteins phloem physiology protein targeting potato.

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The Potato Sucrose Transporter StSUT1 Interacts with a DRM-Associated Protein Disulfide Isomerase

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Molecular Plant 2012年第1期5卷 43-62页

作者： Undine Krugel Hong-Xia He Konstanze Gier Jana Reins Izabela Chincinska Bernhard Grimm Waltraud X. Schulze christina kuhn Institute of Biology Department of Plant Physiology Humboldt University 10115 Berlin Germany Max Planck Institute of Molecular Plant Physiology Am MGhlenberg 1 14476 Potsdam-Golm Germany

Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUTl-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane （DRM） fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiq- uitin system （SUS） to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane- associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUTl-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was ob- served between the StSUTl-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUTl-interacting proteins, a protein disulfide isomerase （PDI）, interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

关键词： Protein-protein interactions phloem proteins vesicle trafficking endocytosis.

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Beta-7 integrin controls enterocyte migration in the small intestine

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World Journal of Gastroenterology 2015年第6期21卷 1759-1764页

作者： Elke Kaemmerer Paula kuhn Ursula Schneider Thomas Clahsen Min Kyung Jeon christina Klaus Julia Andruszkow Michael Hrer Sabine Ernst Angela Schippers Norbert Wagner Nikolaus Gassler Institute of Pathology RWTH Aachen University Department of Pediatrics RWTH Aachen University Department of Medical Statistics RWTH Aachen University

AIM:To hypothesize that beta-7 integrin affects cellularmigration of both,lymphocytes and ***:The nucleoside analog Brd U was ip injected in beta-7-deficient mice(C57BL/6-Itgbtmlcgn/J)of male gender and age-matched male C57BL/J J mice(wild type)4,20,or 40 h before *** total small intestine was isolated,dissected,and used for morphometrical *** U-positive epithelial cells were numbered in at least 15 hemi-crypts per duodenum,jejunum,and ileum of each *** outer most Brd U-positive cell(cellmax)was determined per hemi-crypt,numerically documented,and statistically ***:Integrins containing the beta-7-chain were exclusively expressed on *** the small intestinal mucosa of beta-7 integrin-deficient mice the number of intraepithelial lymphocytes was drastically ***,the Peyer’s patches of beta-7integrin-deficient mice appeared *** beta-7integrin-deficient mice the location of cellmax was found in a higher position than it was the case for the *** difference was already detected at 4 h after Brd U application,but significantly increased with time(40 h after Brd U injection)in all small intestinal segments investigated,i.e.,duodenum,jejunum,and *** of small intestinal enterocytes was different between the experimental groups measured by cellmax ***:The E-cadherin beta-7 integrin pathway probably controls migration of enterocytes within the small intestinal surface lining epithelial layer.

关键词： Barrier function Cell migration Inflammatory bowel

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Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes

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World Journal of Gastroenterology 2015年第15期21卷 4499-4508页

作者： Elke Kaemmerer Paula kuhn Ursula Schneider Min Kyung Jeon christina Klaus Miriam Schiffer Danika Weisner Christian Liedtke J?rg J?kel Lieven Nils Kennes Ralf-Dieter Hilgers Norbert Wagner Nikolaus Gassler Institute of Pathology RWTH Aachen University Department of Pediatrics RWTH Aachen University Department of Medicine Ⅲ RWTH Aachen University Department of Medical Statistics RWTH Aachen University

AIM: To verify the hypothesis that caspase-8(Casp8), which regulates cellular apoptosis and necroptosis, is critically involved in enterocyte ***: Casp8-silenced Caco2 cells were used in migration assays. In addition, enterocyte-specific Casp8 heterozygous(Casp8+/?int) or homozygous knockout mice(Casp8?int) were generated by crossing genetically modified mice carrying lox P recombination sites in intron 2 and 4 of the murine Casp8 gene with transgenic animals expressing a cre-transgene under control of the villin promoter in a pure C57/BL6 genetic background. The nucleoside analog Brd U was injected i.p. in male Casp8+/?int and Casp8?int animals 4 h, 20 h, or 40 h before performing morphometric studies. Locations of anti-Brd U-immunostained cells(cellmax) in at least 50 hemi-crypts of 6 histoanatomically distinct intestinal mucosal regions were numbered and extracted for statistical procedures. For the mice cohort(n = 28), the walking distance of enterocytes was evaluated from cellmax within crypt(n = 57), plateau(n = 19), and villus(n = 172) positions, resulting in a total of 6838 observations. Data analysis was performed by fitting a three-level mixed effects modelto the ***: In cell culture experiments with Caco2 cells, Casp8 knockdown efficiency mediated by RNA interference on Casp8 transcripts was 80% controlled as determined by Western blotting. In the scratch assay, migration of Casp8-deleted Caco2 cells was significantly diminished when compared with controls(Casp8?scramble and Caco2). In Brd U-labeled Casp8?int mice, cellmax locations were found along the hemi-crypts in a lower position than it was for Casp8+/?int or control(cre-negative) animals. Statistical data analysis with a three-level mixed effects model revealed that in the six different intestinal locations(distinct segments of the small and large intestine), cell movement between the three mice groups differed widely. Especially in duodenal hemi-crypts, enterocyte movement w

关键词： Barrier function Caspase 8 Cell migration Inflammatory bowel disease Intestinal morphogenesis

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