Brian K. Sato

Misfolded Membrane Proteins Are Specifically Recognized by the Transmembrane Domain of the Hrd1p Ubiquitin Ligase

Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multispanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intramembrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients.

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

A screen for GFP-tagged yeast proteins that can assemble into visible structures reveals four new filamentous structures in the cytoplasm formed by metabolic enzymes and translation factors.

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process.

Yeast Derlin Dfm1 interacts with Cdc48 and functions in ER homeostasis

Recent studies have identified Derlin‐1, a protein that associates with the AAA‐ATPase p97 and is implicated in late steps in ER‐associated protein degradation (ERAD). Derlin‐1 has two Saccharomyces cerevisiae homologues, Der1p and Dfm1p. While Der1p has been studied extensively, little is known about Dfm1p. Accordingly, we investigated the role of Dfm1p in ERAD, ER homeostasis and interactions with the yeast p97 homologue Cdc48p. Dfm1p was not involved in the degradation of a number of Der1‐dependent or ‐independent ERAD substrates, neither was it redundant with either Der1p or Sec61p in ERAD. However, Dfm1p had a role in ER homeostasis, since Dfm1p loss or overexpression could stimulate the unfolded protein response (UPR). Furthermore, Dfm1p interacted both genetically and physically with Cdc48p, the yeast p97 homologue, and this interaction required an eight amino acid sequence found in the C‐terminus of Dfm1p that we have termed the SHP box. Our genetic studies are consistent with the lack of a role for Dfm1p in ERAD, but indicate it participates in ER‐related Cdc48p actions distinct from retrotranslocation. Finally, sequence analysis indicated that the UPR‐related and Cdc48p interaction functions of Dfm1p could be separated, implying this protein probably has numerous actions in the cell. Thus, the interaction between Derlins and p97 is conserved between yeast and mammals, although its function in ERAD is not. Furthermore, Dfm1p interacts with Cdc48p through its SHP boxes, and so defines a new motif for interaction with this widely‐employed AAA‐ATPase. Copyright

Practice Makes Pretty Good: Assessment of Primary Literature Reading Abilities across Multiple Large-Enrollment Biology Laboratory Courses

Incorporation of a module focused on primary literature into three upper-division biology lab courses resulted in biology discipline–independent longitudinal learning gains for enrolled undergraduates. This module is easily transferable and is modeled around the principles used by researchers when approaching a scientific paper.

Brewing for Students: An Inquiry-Based Microbiology Lab

In an effort to improve and assess student learning, there has been a push to increase the incorporation of discovery-driven modules and those that contain real-world relevance into laboratory curricula. To further this effort, we have developed, implemented, and assessed an undergraduate microbiology laboratory experiment that requires students to use the scientific method while brewing beer. The experiment allows students to brew their own beer and characterize it based on taste, alcohol content, calorie content, pH, and standard reference method. In addition, we assessed whether students were capable of achieving the module learning objectives through a pre-/posttest, student self-evaluation, exam-embedded questions, and an associated worksheet. These objectives included describing the role of the brewing ingredients and predicting how altering the ingredients would affect the characteristics of the beer, amongst others. By completing this experimental module, students accomplished the module objectives, had greater interest in brewing, and were more likely to view beer in scientific terms. Journal of Microbiology & Biology Education.

A Familiar(ity) Problem: Assessing the Impact of Prerequisites and Content Familiarity on Student Learning.

Prerequisites are embedded in most STEM curricula. However, the assumption that the content presented in these courses will improve learning in later courses has not been verified. Because a direct comparison of performance between students with and without required prerequisites is logistically difficult to arrange in a randomized fashion, we developed a novel familiarity scale, and used this to determine whether concepts introduced in a prerequisite course improved student learning in a later course (in two biology disciplines). Exam questions in the latter courses were classified into three categories, based on the degree to which the tested concept had been taught in the prerequisite course. If content familiarity mattered, it would be expected that exam scores on topics covered in the prerequisite would be higher than scores on novel topics. We found this to be partially true for “Very Familiar” questions (concepts covered in depth in the prerequisite). However, scores for concepts only briefly discussed in the prerequisite (“Familiar”) were indistinguishable from performance on topics that were “Not Familiar” (concepts only taught in the later course). These results imply that merely “covering” topics in a prerequisite course does not result in improved future performance, and that some topics may be able to removed from a course thereby freeing up class time. Our results may therefore support the implementation of student-centered teaching methods such as active learning, as the time-intensive nature of active learning has been cited as a barrier to its adoption. In addition, we propose that our familiarity system could be broadly utilized to aid in the assessment of the effectiveness of prerequisites.

The grass isn’t always greener: Perceptions of and performance on open-note exams

The performance and perception of open- and closed-note testing environments were investigated. Exam scores did not vary under these conditions, but small differences were uncovered for students with experience taking open-note exams. This implies that increased exposure to open-note testing is required to understand the impact of this intervention.

How do students study in STEM courses? Findings from a light-touch intervention and its relevance for underrepresented students

With the nationwide emphasis on improving outcomes for STEM undergraduates, it is important that we not only focus on modifying classroom instruction, but also provide students with the tools to maximize their independent learning time. There has been considerable work in laboratory settings examining two beneficial practices for enhancing learning: spacing and self-testing. In the current study, we examine biology students’ study practices, particularly in the context of these two behaviors. We specifically investigate whether a light-touch study skills intervention focused on encouraging spacing and self-testing practices impacted their utilization. Based on pre- and post-course surveys, we found that students report utilizing both beneficial and ineffective study practices and confirm that usage of spacing and self-testing correlates with a higher course grade. We also found that students in the section of the course which received the study skills intervention were more likely to report continued use or adoption of spacing and self-testing compared to students in control sections without the intervention. Surprisingly, we found that underrepresented minorities (URMs) under-utilize self-testing, and that our intervention helped to partially ameliorate this gap. Additionally, we found that URMs who reported self-testing earned similar course grades compared to non-URMs who also self-tested, but that there was a much larger drop in performance for URMs who did not self-test relative to non-URMs who also did not self-test. Overall, we would encourage instructors to dedicate class time towards discussing the merits of beneficial study practices, especially for students that have historically underperformed in STEM disciplines.

The Impact of Instructor Exam Feedback on Student Understanding in a Large-Enrollment Biology Course

Encouraging students to reflect on their exam answers in response to expert feedback may result in increased classroom performance and highlights that learning is an ongoing experience rather than one terminated at exam completion. In this study, we assessed the impact of instructor-delivered midterm feedback, employing a system in which students were not only given the opportunity to reflect on their answers, but to then engage in an email discussion about the feedback with the instructor. We found that the students who participated in the feedback process provided higher quality responses to similar final-exam questions. Using surveys and interviews, we identified components of the feedback process that the students perceived to be most valuable. We would encourage instructors to incorporate exam feedback in the assessment process not only because of its potential to enhance learning but also to increase opportunities for students to reflect on their own understanding.