Domains of a Transit Sequence Required for in Vivo Import in Arabidopsis Chloroplasts1

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.

Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.

Mobility and Aging in Denver, Colorado: Travel Behavior, Mobility Barriers, and Perceptions of Transit

The number of seniors in the U.S. today is growing rapidly because of longer life expectancies and the aging Baby Boomer generation. This age groups' travel behavior will have substantial impacts on transportation, economics, safety, and the environment. This research used a mixed-methods approach to address issues of mobility and aging in Denver, Colorado. A quantitative approach was used to answer broad questions about travel behavior and the effects of age, gender, work status, disability, residential location and socio-economic status on mobility. Qualitative interviews with seniors in the Denver metro area were conducted to identify barriers to mobility, decision-making processes and travel decisions, and seniors' perceptions of public transit. The results of the quantitative and qualitative analyses show that residential location is an important variable for determining seniors' travel behaviors and transportation options. Perceptions of public transit were positive, but accessibility and information barriers exist that prevent older adult from using transit. The findings of this study will help to provide transportation and service recommendations to policymakers and planners in the Denver area as well as to inform studies of other North American cities with large aging populations.

The Impact of Transit Oriented Development on the Travel Behaviors of Workers in Denver, Colorado

To combat unsustainable transportation systems characterized by reliance on petroleum, polluting emissions, traffic congestion and suburban sprawl, planners encourage mixed use, densely populated areas that provide individuals with opportunities to live, work, eat and shop without necessarily having to drive private automobiles to accommodate their needs. Despite these attempts, the frequency and duration of automobile trips has consistently increased in the United States throughout past decades. While many studies have focused on how residential proximity to transit influences travel behavior, the effect of workplace location has largely been ignored. This paper asks, does working near a TOD influence the travel behaviors of workers differently than workers living near a TOD? We examine the non-work travel behaviors of workers based upon their commuting mode and proximity to TODs. The data came from a 2009 travel behavior survey by the Denver Regional Council of Governments, which contains 8,000 households, 16,000 individuals, and nearly 80,000 trips. We measure sustainable travel behaviors as reduced mileage, reduced number of trips, and increased use of non-automobile transportation. The results of this study indicate that closer proximity of both households and workplaces to TODs decrease levels of car commuting and that non-car commuting leads to more sustainable personal travel behaviors characterized by more trips made with alternative modes.

Opportunities and Challenges for the Widespread Implementation of Battery Powered Electric Transit Buses

The need for more sustainable public transportation choices drives innovation and provides opportunity for improvement in options. Transit buses provide many advantages for efficient transportation and electric drive vehicles are anticipated to play an increasing role in future transportation systems. A lifecycle cost analysis of battery electric transit buses indicates rate structures and demand charges do not currently have a large impact on lifecycle cost for small fleets of battery electric buses. As fleets grow, policies and rate structures will need to adjust to avoid becoming a barrier to adoption. Battery electric transit buses are now being developed which promise to address the primary issues of high life cycle cost, low reliability, range, and flexibility.

Entre économie de la fonctionnalité et monopole : la United Shoe Machinery Company dans la première moitié du XXe siècle

L’histoire de la United Shoe Machinery Company (USMC) montre que la réalité ne s’adapte pas toujours aux simplifications de la théorie. Comme le signale l’économie de la fonctionnalité, la stratégie de l’entreprise de vendre l’usage plutôt que la machine fournit plusieurs avantages importants, mais elle a également contribué au fait que les usines de chaussures subissent une véritable dépendance technologique de cette compagnie et au fait que l’USMC soit parvenue à une domination monopolistique du marché. D’autre part, en remettant en cause les rudiments généraux de la théorie économique néoclassique, cette position de monopole n’a pas empêché que l’entreprise ait un fonctionnement efficace et ait facilité la modernisation technologique de l’industrie de la chaussure, aux États-Unis et dans d’autres pays.

Company orders, 1810 May 2

A printed request for Croswell to appear for military duty.