Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

Background

Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results

In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion

A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

     

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

Background  

Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine.     

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.     

Endogenous estradiol and testosterone levels are associated with cognitive performance in older women and men

Relatively few studies have investigated the relationship between endogenous sex steroid levels and cognition in older people and the reported results have been inconsistent. A number of experimental hormone replacement studies have suggested that estrogen replacement in older women enhances cognition, especially verbal memory. In contrast, little research has been done focusing on men. In the current study the association between endogenous sex steroids (estradiol and testosterone) and cognition was investigated in 38 healthy older women (mean age 68 years) and 30 healthy older men (mean age 69 years). Five cognitive tests measuring verbal memory, spatial memory, verbal fluency, mental rotation, and susceptibility to interference were administered. Results revealed that in women higher estradiol levels as well as testosterone levels were associated with better verbal memory (paired associates and estradiol; r =.38, P < 0.05; paired associates and testosterone; r =.33, P < 0.05;). Moreover estradiol, but not testosterone was associated with less susceptibility to interference (Stroop color word test; r = -0.34, P < 0.05). In men the only significant association was a negative correlation between testosterone and verbal fluency (r = -0.38, P < 0.05). The associations observed in this small study support the notion that estradiol is protecting verbal memory and possibly also frontal lobe mediated functions in older women. In contrast to the positive findings in women endogenous sex steroids do not appear to be closely linked to better cognition in older men.     

A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy

Tripeptidyl peptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases that is thought to act downstream of the proteasome in the ubiquitin-proteasome pathway. Recently, a key role in a pathway parallel to the ubiquitin-proteasome pathway has been ascribed to TPP II, which forms a giant protease complex in mammalian cells. Here, we report the 900-fold purification of TPP II from Drosophila eggs and demonstrate via cryo-electron microscopy that TPP II from Drosophila melanogaster also forms a giant protease complex. The presented three-dimensional reconstruction of the 57 x 27 nm TPP II complex at 3.3 nm resolution reveals that the 150 kDa subunits form a superstructure composed of two segmented and twisted strands. Each strand is 12.5 nm in width and composed of 11 segments that enclose a central channel.     

RHIZOME AND ROOT GROWTH RATES AND CYCLES IN PROTEIN AND CARBOHYDRATE CONCENTRATIONS IN GEORGIA SPARTINA ALTERNIFLORA LOISEL. PLANTS

Root and rhizome growth and composition were measured in a Georgia salt marsh. Root growth was less seasonal than rhizome growth; none of the latter occurred during the first quarter of the year. The depth of underground growth shifted downward during the fall. Sugar and starch concentrations were relatively low in roots throughout the year. They remained relatively high in the rhizomes, but varied in distribution depending on the season.     

Reproductive inefficiency in male black-footed ferrets (Mustela nigripes)

The black-footed ferret (Mustela nigripes), once considered extinct, has benefited from captive breeding and subsequent reintroduction into native habitat. A high proportion of females (>90%) exhibit estrus in captivity during the spring breeding season. However, many males considered to be prime-breeding age (1-3 years old) fail to sire offspring. Breeding records in 1995 revealed that 40 of 73 males (55%) managed under the Black-Footed Ferret Species Survival Plan did not reproduce, despite being provided opportunity. The present study was conducted to determine the incidence and etiology of male reproductive failure in 1996 and 1997. In 1996, 38 of 69 (55%) 1- to 3-year-old males failed to sire offspring. Likewise, 35 of 60 (58%) males did not reproduce in 1997. Overall, 21% of adult males failed to sire young in three consecutive breeding seasons (1995-1997). Electroejaculate traits (ejaculate volume, sperm concentration, motility, morphology, and acrosomal integrity) from 29 proven breeder males were not different (P > 0.05) from those of 23 males that did not sire young (nonproven breeders). However, six categories of reproductive failure were identified for the 73 prime-breeding age, nonproven males: 1) underdeveloped testes (22%); 2) improper breeding position with the female (25%); 3) excessive aggression toward estrous females (9%); 4) copulation with no sperm detected at postcoital lavage (19%); 5) copulation with sperm in the vaginal lavage but no resulting pregnancy (18%); and 6) copulation with no vaginal lavage performed and no resulting pregnancy (7%). These data indicate that combined behavioral and physiologic factors, but not overall sperm quality, influence reproductive performance in male black-footed ferrets managed in captivity. Zoo Biol 19:517-528, 2000. Copyright 2000 Wiley-Liss, Inc.