Location is everything: an educational primer for use with "genetic analysis of the ribosome biogenesis factor Ltv1 of Saccharomyces cerevisiae"

Abstract

The article by Merwin et al. in the November 2014 issue of GENETICS provides insight into ribosome biogenesis, an essential multistep process that involves myriad factors and three cellular compartments. The specific protein of interest in this study is low-temperature viability protein (Ltv1), which functions as a small ribosomal subunit maturation factor. The authors investigated its possible additional function in small-subunit nuclear export. This Primer provides information for students to help them analyze the paper by Merwin et al. (2014), including an overview of the authors' research question and methods.

Keywords: education; nuclear export; ribosome biogenesis.

Figure 1

Pathway for maturation of pre-ribosomes to form 40S and 60S ribosomal subunits. Sequential assembly intermediates are shown, distinguished by the pre-rRNA processing intermediates contained within them. Most r-proteins (light blue) and many assembly factors (dark blue) associate with the early nucleolar/nuclear precursor particles. Some assembly factors join pre-ribosomes in midassembly or even during later steps in the cytoplasm. Release of assembly factors from pre-ribosomes occurs at early, middle, or late stages of subunit maturation (from Woolford and Baserga 2013).

Figure 2

Export of the pre-60S ribosomal subunit requires an export adapter, Crm1, and Ran-GTP. Many protein factors () are associated with the pre-60S ribosomal subunit in the nucleus. The export adapter () provides a link between the pre-60S subunit and the Crm1 export complex (). Ran-GTP () is bound to the Crm1 export complex and, after export from the nucleus, is hydrolyzed to Ran-GDP, allowing release of the 60S ribosomal complex.

Figure 3

Sucrose density gradient centrifugation. The gradient is from 10% sucrose at the top to 50% at the bottom. LMW, low molecular weight; HMW, high molecular weight. Whole-cell lysates are prepared and layered carefully on top of the gradient. As indicated, individual ribosomal subunits or the complete ribosome is less dense than polysomes and therefore ends up near the top of the gradient after centrifugation (from Abdelmohsen 2012).

Figure 4

The yeast two-hybrid system. A. (Left) Representation of the yeast Gal4 transcription activator with the DNA-binding (GBD) and transcription-activation (GAD) domains colored in different shades of blue, as indicated. (Right) Representations of two hybrid proteins with Ltv1 fused to the Gal4 activation domain (GAD) and Crm1 fused to the Gal4 DNA-binding domain (GBD). B. (Top) Hypothetical scenario in which Ltv1 and Crm1 interact, leading to expression of ADE2 and (bottom) hypothetical scenario in which Ltv1 and Crm1 do not interact (adapted from Duina et al. 2014 and James et al. 1996).