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Copper uptake by CMC1 Deletion in

Saccharomyces Cerevisiae

Nick Corbett, Scott Voorhees and Daren Heaton

Utah Valley University, 800 W University Parkway, Orem Utah 84058.

Abstract

Introduction

Materials and Methods Flame Atomic Absorption

Spectroscopy (AAS)

Discussion

References

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Cytochrome c oxidase (CcO) or complex IV is the

terminal component of the electron transport chain. In

eukaryotic organisms, CcO is composed of 12-13

subunits. The core of eukaryotic CcO contains three

mitochondrially encoded subunits that comprise the

catalytic core of the complex and several gene products

encoded for by the nucleus (1). Essential to the redox

function of CcO are several critical cofactors: two

hemes and two copper centers (2). The crystal

structure of CcO has led us to several insights about its

structural components and catalytic activity (3).

However, a large set of nuclear gene products are

essential for CcO activity that are not part of the

structural machinery of the complex (4,5). These

components have been implicated in various stages of

CcO assembly including, heme processing and

insertion (6), Cu

and Cu

site delivery and insertion

(7), subunit processing and subunit assembly (8,9).

Among them are well characterized CcO assembly

factors that involve the biogenesis of the Cu

and Cu

sites in CcO. Sco I, Cox17, Cox 11 and Cox23 are all

essential to CcO activity and have properties that

appear to be critical to in the maturation of the Cu

and

sites(10-13). Cmc1 has also been implicated in

CcO activity. Cmc1 may have an important role in

copper sorting within the mitochondria (14).

1. Capaldi. R.A. Structure and function of cytochrome c oxidase. Annu. Rev. Biochem 1990, 59:569-596.

2. Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R,

Yaono R, Yoshikawa S. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

Science 1996 272:1136-44.

3. Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-Itoh K, Nakashima R,

Yaono R, Yoshikawa S. Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8

A, Science 1995, 269:1069-74.

4. J.E. McEwen, C. Ko, B. Kloeckner-Griussem, R.O. Poyton. Nuclear functions required for

cytochrome c oxidase biogensis in Saccharomyces cerevisiae. Characterization of mutants in 34

complementation groups. J Biol. Chem. 1987 261:11872-11879.

5. A Tzagoloff, C.L. Dieckmann, PET genes of Saccharomyces cerevisiae, Microbiol. Rev. 1990 54:211-

225.

6. Khalimonchuk O, Bestwick M, Meunier B, Watts TC, Winge DR. Formation of the redox cofactor

centers during Cox1 maturation in yeast cytochrome oxidase. Mol Cell Biol. 2010 30:1004-17.

7. Horng Y.C., Cobine P.A., Maxfield A.B., Carr H.S., Winge D.R. Specific copper transfer from the

Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase. J Biol

Chem. 2004 279:35334-40.

8. Barros MH, Myers AM, Van Driesche S, Tzagoloff A. COX24 codes for a mitochondrial protein

required for processing of the COX1 transcript. J Biol Chem. 2006 281:3743-51.

9. Pierrel F, Khalimonchuk O, Cobine PA, Bestwick M, Winge DR. Coa2 is an assembly factor for yeast

cytochrome c oxidase biogenesis that facilitates the maturation of Cox1. Mol Cell Biol. 2008 16:4927-39.

10.Rigby K, Cobine PA, Khalimonchuk O, Winge DR. Mapping the functional interaction of Sco1 and

Cox2 in cytochrome oxidase biogenesis. J Biol Chem. 2008 283:15015-22.

11.Glerum, D. M., Shtanko, A., and Tzagoloff, A. SCO1 and SCO2 Act as High Copy Suppressors of a

Mitochondrial Copper Recruitment Defect in Saccharomyces cerevisiae J. Biol. Chem. 1996 271:20531–

20535.

12.Barros MH, Johnson A, Tzagoloff A. COX23, a homologue of COX17, is required for cytochrome

oxidase assembly. J Biol Chem. 2004 279:31943-7.

13.Carr HS, Maxfield AB, Horng YC, Winge DR. Functional analysis of the domains in Cox11. J Biol

Chem. 2005 280:22664-9.

14.Darryl Horn, Hassan Al-Ali and Antoni Barrientos. Cmc1p is a Conserved Mitochondrial Twin

C Protein Involved in Cytochrome c Oxidase Biogenesis. Mol. Cell. Biol. 2008 28:4354-4363

15.Crouch S, Holler, F. Skoog, D. (2007). Principles of Instrumental Analysis 6

Edition. Belmont: Thomson

Brooks/Cole. 230-250.

The CMC1 deletions appears to uptake less copper

when media is supplement with .04% copper

sulfate or higher.

Results

MATERIALS AND METHODS

A 20% glucose solution and minimal media solution (5 g of

ammonium sulfate, 1.7 g of yeast nitrogen base without

amino acids, 0.73 g of amino acid dropout mix (CSM-ADE-

TRP)) were made. Poured 45 mL of minimal media into

separate 250 mL Erlenmeyer flasks and then autoclaved the

minimal media and the 20% glucose solutions. After the

solutions cooled down, added 5 mL of the 20% glucose

solution to the Erlenmeyer flasks.

Inoculated one of the 250 mL Erlenmeyer flasks with the

wild type cells and another flask with the CMC1 cells and

allowed them to grow for less than 24 hours in a 30°C

incubator rotating at 250 rpm. Once OD reached 0.3 (1:10

dilution)—UV-Vis spectrometer measurement—transferred

the calculated amount of cells to the shock series flasks and

allowed to grow for less than 24 hours. Once OD reached

0.3 (1:10 dilution)—UV-Vis spectrometer measurement—

from the shock series flasks, the corresponding copper

percent was added and the shock series started with 15

minute 1 mL samples taken for up to two hours (0.5 M Cu

solution was prepared from 19.95 g of CuSO

in a 250 mL

volumetric flask).

The 1 mL samples were centrifuged in 2 mL micro

centrifuge tubes twice with the second centrifuge done with

the removal of the supernatant. Removed 0.5 mL of these

different cell solutions, added them to separate appropriately

labeled 50 mL volumetric flasks, and then filled to the mark

of the flask with 2% nitric acid. Transferred these solutions

to appropriately labeled 50 mL conical tubes to be run on the

AAS.

Made 2% nitric acid solutions and made 0.5 ppm, 1 ppm, 1.5

ppm, 2 ppm, 2.5 ppm, and 3 ppm Cu standards (The blank

was the 2% nitric acid solution with no Cu added). After

calibrating the AAS, the samples were run.

Discrepancies in the amount of Cu uptake between the two (CMC1 &

Wild), over the 2hr time period, was only observed at the upper limit

of the Cu% (0.04% and 0.05% Cu) added. Though the 0.04% Cu

series only showed minor differences in the amount of Cu uptake the

trend drastically increased at the 0.05% Cu series.

Troubleshooting Flame AAS:

• Mirrors and Lenses aligned correctly for optimal incidence.

• Nebulizer optimized

• Burner height optimized for optimal incidence at the correct

flame temperature.

The discrepancy in the uptake of Cu at the two upper limits (0.04%

& 0.05% Cu), in the Cu concentration series, may be attributed to

the buildup of a Cu concentration within the cytoplasm of the cell.

Other factors play a role in the high Cu limit of transporting Cu out

of the cytoplasm, due to the mitochondria’s inability to uptake

copper in the CMC1 cells. Comparison of the wild type suggests

that the wild type cells, at the two upper limits of the Cu

concentration series, do not invoke this mechanism due to the lack

of buildup of Cu within the cytoplasm as the available transport

mechanism of Cu into the mitochondria is available.

This mechanism provides and explanation as to the presence of a

higher Cu concentration outside of the CMC1 cells at the two upper

limits of copper concentration. The validity of the mechanism

requires that a similar analysis should be done to measure the

amount of Cu within the cytoplasm.