Jinnie M. Garrett

Mft52, an Acid-bristle Protein in the Cytosol That Delivers Precursor Proteins to Yeast Mitochondria

We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal “acid-bristle” domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.

MITOCHONDRIAL PROTEIN IMPORT : ISOLATION AND CHARACTERIZATION OF THE SACCHAROMYCES CEREVISIAE MFT1 GENE

SummaryMitochondrial targeting of an Atp2-LacZ fusion protein confers a respiration-defective phenotype on yeast cells. This effect has been utilized to select strains that grow on nonfermentable carbon sources, some of which have decreased levels of hybrid protein localized to the organelle. Many of the mutants obtained were also temperature-sensitive for growth on all media. The recessive mft (mitochondrial fusion targeting) mutants have been assigned to three complementation groups. MFT1 was cloned and sequenced: it encodes a 255 amino acid protein that is highly basic and has no predicted membrane-spanning domains or organelle-targeting sequences. The MFT1 gene is 91% identical to an open reading frame 3′ of the SIR3 gene. Evidence is presented that these two closely related genes could represent a recent gene duplication.

The control of morphogenesis in Saccharomyces cerevisiae by Elm1 kinase is responsive to RAS/cAMP pathway activity and tryptophan availability

Many fungi undergo a morphological transition to filamentous growth in response to limiting nutrient conditions. Constitutively elongated Saccharomyces cerevisiae mutants (elm) have been isolated; the ELM1 gene encodes a putative serine/threonine protein kinase. A novel allele, elm1‐15, has been isolated in an S288C‐derived strain, which causes a pleiotropic phenotype, including media‐specific growth effects, abnormal morphology and altered stress response, in cells that are auxotrophic for tryptophan. elm1‐15 trp1 cells cannot use many nitrogen sources, are sensitive to amino acid analogues, have very low general amino acid permease activity and do not accumulate trehalose. In contrast, haploid elm1‐15 TRP1 cells grow well in budding form on all media, are stress resistant and overaccumulate trehalose. Several lines of evidence suggest that Elm1 acts on functions related to the RAS/cAMP pathway. Overexpression of Elm1 partially rescues the ts phenotype of cdc25 and cyr1 mutants. Deletion of ELM1 in low PKA activity mutants increased the severity of their phenotypes, and activation of Ras2 decreases the cell elongation phenotype of elm1 mutants. A ‘signal integration’ model for the complex relationship of Elm1 and the RAS/cAMP pathway in controlling morphogenesis in response to nutrients is proposed.

The Branched-Chain Amino Acid Permease Gene of Saccharomyces cerevisiae, BAP2, Encodes the High-Affinity Leucine Permease (S1)

The amino acid leucine has been shown previously to be transported into a yeast cell by at least three permeases: the general amino acid permease, a high‐affinity permease (S1) and a low‐affinity permease (S2). We isolated the gene BAP2 as a multicopy suppressor of the YPD− phenotype of aat1leu2 yeast. BAP2 has been identified previously as encoding an amino acid permease which transports branched‐chain amino acids. In order to align the genetic and biochemical studies of leucine uptake we completed a detailed kinetic analysis of yeast strains in which the BAP2 gene was disrupted and compared this to the kinetics of uptake of the parental strain. We demonstrate that BAP2 encodes the high‐affinity leucine permease previously called S1.

Chapter 2 Resources and Strategies to Integrate the Study of Ethical, Legal, and Social Implications of Genetics into the Undergraduate Curriculum

Gene therapy, genetically modified organisms, and the privacy of an individuals genetic information are just a few of the developments emerging from recent advances in molecular genetics that are controversial. Oversight and regulation of emerging technologies are the responsibility of both experts and the general public who both need to understand the science and the societal impact of its use. The study of ethical, legal, and social implications (ELSI) of advances in genetics provides a very powerful pedagogical tool to accomplish two goals. These are, first of all, to interest nonscientists in genetics and engage them in learning the science behind the ELSI developments they are considering, and secondly, to broaden the perspective of science students to consider the history and social consequences of the science they are studying. The resources and strategies presented in this chapter for teaching ELSI issues that arise in modern genetics are designed to aid in accomplishing these goals throughout the undergraduate curriculum. This chapter provides (1) a set of nine ELSI topic modules that can be incorporated into courses for both majors (from introductory to graduate level) and nonmajors and (2) examples of course pedagogy for specific classes.

A low cost multi-level sampling device for synchronous aseptic collection of environmental water samples

We describe a simple device for the aseptic collection of environmental water samples at high spatial resolution to depths of 50m. To demonstrate the utility of this technique we present geochemical and archaeal community data from samples collected throughout the water column of a stratified lake.