Christine Pfund

Getting Newly Synthesized Proteins into Shape

Recent progress made in the analysis of in vivo folding of cytosolic proteins suggests that folding of cytosolic proteins occurs via multiple chaperone-assisted, as well as unassisted pathways. In the case of individual proteins, certain pathways may be highly favored. But, the cellular folding machinery also shows significant redundancy and flexibility, resulting in a variable network of folding pathways having alternative routes and backup systems.‡To whom correspondence should be addressed (e-mail: ecraig@facstaff.wisc.edu [E. A. C.], bukau@sun2.ruf.uni-freiburg.de [B. B.]).

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome–nascent chain complex

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb–ribosome interaction was characterized. Incorporation of the aminoacyl‐tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross‐linked to nascent chains containing a modified lysine residue with a photoactivatable cross‐linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome–nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Within-Species Flagellin Polymorphism in Xanthomonas campestris pv campestris and Its Impact on Elicitation of Arabidopsis FLAGELLIN SENSING2–Dependent Defenses

Bacterial flagellins have been portrayed as a relatively invariant pathogen-associated molecular pattern. We have found within-species, within-pathovar variation for defense-eliciting activity of flagellins among Xanthomonas campestris pv campestris (Xcc) strains. Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a transmembrane leucine-rich repeat kinase, confers flagellin responsiveness. The flg22 region was the only Xcc flagellin region responsible for detectable elicitation of Arabidopsis defense responses. A Val-43/Asp polymorphism determined the eliciting/noneliciting nature of Xcc flagellins (structural gene fliC). Arabidopsis detected flagellins carrying Asp-43 or Asn-43 but not Val-43 or Ala-43, and it responded minimally for Glu-43. Wild-type Xcc strains carrying nonrecognized flagellin were more virulent than those carrying a recognized flagellin when infiltrated into Arabidopsis leaf mesophyll, but this correlation was misleading. Isogenic Xcc fliC gene replacement strains expressing eliciting or noneliciting flagellins grew similarly, both in leaf mesophyll and in hydathode/vascular colonization assays. The plant FLS2 genotype also had no detectable effect on disease outcome when previously untreated plants were infected by Xcc. However, resistance against Xcc was enhanced if FLS2-dependent responses were elicited 1 d before Xcc infection. Prior immunization was not required for FLS2-dependent restriction of Pseudomonas syringae pv tomato. We conclude that plant immune systems do not uniformly detect all flagellins of a particular pathogen species and that Xcc can evade Arabidopsis FLS2-mediated defenses unless the FLS2 system has been activated by previous infections.

Zuotin, a ribosome‐associated DnaJ molecular chaperone

Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins. In Saccharomyces cerevisiae, SSB proteins are ribosome‐associated Hsp70s which interact with the newly synthesized nascent polypeptide chain. Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ‐related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity. Zuo1, which has been shown previously to be a nucleic acid‐binding protein, is also a ribosome‐associated protein localized predominantly in the cytosol. Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA. We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome.

Flagellin Is Not a Major Defense Elicitor in Ralstonia solanacearum Cells or Extracts Applied to Arabidopsis thaliana

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.

Novel G-Protein Complex Whose Requirement Is Linked to the Translational Status of the Cell

ABSTRACT G proteins, which bind and hydrolyze GTP, are involved in regulating a variety of critical cellular processes, including the process of protein synthesis. Many members of the subfamily of elongation factor class G proteins interact with the ribosome and function to regulate discrete steps during the process of protein synthesis. Despite sequence similarity to factors involved in translation, a role for the yeast Hbs1 protein has not been defined. In this work we have identified a genetic relationship between genes encoding components of the translational apparatus and HBS1. HBS1, while not essential for viability, is important for efficient growth and protein synthesis under conditions of limiting translation initiation. The identification of an Hbs1p-interacting factor, Dom34p, which shares a similar genetic relationship with components of the translational apparatus, suggests that Hbs1p and Dom34p may function as part of a complex that facilitates gene expression. Dom34p contains an RNA binding motif present in several ribosomal proteins and factors that regulate translation of specific mRNAs. Thus, Hbs1p and Dom34p may function together to help directly or indirectly facilitate the expression either of specific mRNAs or under certain cellular conditions.

Training Mentors of Clinical and Translational Research Scholars: A Randomized Controlled Trial

Purpose To determine whether a structured mentoring curriculum improves research mentoring skills. Method The authors conducted a randomized controlled trial (RCT) at 16 academic health centers (June 2010 to July 2011). Faculty mentors of trainees who were conducting clinical/translational research ≥50% of the time were eligible. The intervention was an eight-hour, case-based curriculum focused on six mentoring competencies. The primary outcome was the change in mentors’ self-reported pretest to posttest composite scores on the Mentoring Competency Assessment (MCA). Secondary outcomes included changes in the following: mentors’ awareness as measured by their self-reported retrospective change in MCA scores, mentees’ ratings of their mentors’ competency as measured by MCA scores, and mentoring behaviors as reported by mentors and their mentees. Results A total of 283 mentor–mentee pairs were enrolled: 144 mentors were randomized to the intervention; 139 to the control condition. Self-reported pre-/posttest change in MCA composite scores was higher for mentors in the intervention group compared with controls (P < .001). Retrospective changes in MCA composite scores between the two groups were even greater, and extended to all six subscale scores (P < .001). More intervention-group mentors reported changes in their mentoring practices than control mentors (P < .001). Mentees working with intervention-group mentors reported larger changes in retrospective MCA pre-/posttest scores (P = .003) and more changes in their mentors’ behavior (P = .002) than those paired with control mentors. Conclusions This RCT demonstrates that a competency-based research mentor training program can improve mentors’ skills.

A Research Mentor Training Curriculum for Clinical and Translational Researchers

To design and evaluate a research mentor training curriculum for clinical and translational researchers. The resulting 8‐hour curriculum was implemented as part of a national mentor training trial.

Entering Research: A Course That Creates Community and Structure for Beginning Undergraduate Researchers in the STEM Disciplines

Undergraduate research experiences have been shown to enhance the educational experience and retention of college students, especially those from underrepresented populations. However, many challenges still exist relative to building community among students navigating large institutions. We developed a novel course called Entering Research that creates a learning community to support beginning undergraduate researchers and is designed to parallel the Entering Mentoring course for graduate students, postdocs, and faculty serving as mentors of undergraduate researchers. The course serves as a model that can be easily adapted for use across the science, technology, engineering, and mathematics (STEM) disciplines using a readily available facilitators manual. Course evaluations and rigorous assessment show that the Entering Research course helps students in many ways, including finding a mentor, understanding their place in a research community, and connecting their research to their course work in the biological and physical sciences. Students in the course reported statistically significant gains in their skills, knowledge, and confidence as researchers compared with a control group of students, who also were engaged in undergraduate research but not enrolled in this course. In addition, the faculty and staff members who served as facilitators of the Entering Research course described their experience as rewarding and one they would recommend to their colleagues.

Scientific Teaching in Practice

A new generation of university scientists is learning to teach using a scientific teaching approach.