Sulfhydryl groups on yeast ribosomal proteins L7 and L26 are significantly more reactive in the 80 S particles than in the 60 S subunits.
The sulfhydryl-directed fluorescent reagent, 5-iodoacetamidofluorescein (IAF), reacts differently with proteins from the 60 S ribosomal subunit of Saccharomyces cerevisiae when this subunit is free as opposed to being contained within the 80 S ribosome. When the 80 S ribosomes and the free 60 S subunits were labeled with IAF, the specific fluorescence intensity (fluorescence intensity unit/A260 60 S subunit) of the subsequently derived 60 S was 16.3 and 5.4, respectively. Gel analysis showed that proteins L7 and L26 were selectively labeled and contained greater than 90% of the total fluorescent label, when 80 S ribosomes were labeled. When free 60 S subunits were labeled, six additional proteins were labeled. Both types of modified 60 S subunits were equally capable to support protein synthesis in vitro. Reassociation of the IAF-labeled derived and free 60 S subunits with unmodified 40 S subunits resulted in a maximum of 5-7% decrease and a 3-fold increase, respectively, in the fluorescence intensity without a shift in the emission maxima. The data suggest that ribosomal proteins L7 and L26 contain SH groups that respond to ribosomal subunit association and become more reactive in the intact ribosome than in the subunit. The environments of some or all of the additionally labeled proteins are also sensitive to subunit reassociation.
Lee
J C
JC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Horowitz
P M
PM
D015180:Electrophoresis, Gel, Two-Dimensional; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*
C489218:RPL26A protein, S cerevisiae; C489219:RPL26B protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds