Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain

The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.     

Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.     

Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA

According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.     

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.     

Prevention of flap necrosis by chlorpromazine

Chlorpromazine administered to Sprague-Dawley rats 30 minutes prior to elevation of McFarlane back flaps and continued 14 days thereafter resulted in near complete flap survival, compared with 48 percent necrosis in control animals. Chlorpromazine demonstrates a wide variety of actions that appear to meet all presently known requirements for flap preservation.