RESEARCH TOPICS

Cytochrome c assembly (Overview)

Cytochrome c assembly (Overview)

Since 1987 our group has studied the molecular mechanisms by which the three pathways (systems I, II, III) covalently attach heme to the protein (apocytochrome c). These systems are diagrammed on the Home page.  We use a combination of molecular genetics and membrane protein biochemistry to elucidate, for example, how heme moves through each system and is ultimately attached. Some of the tools we employ and details on assembly are described below.  

We are also interested in finding chemical inhibitors of these pathways, since systems I and II are used by prokaryotes, yet humans use the simple, unrelated system III.  We discovered that metal (non-iron) porphyrins specifically inhibit systems I and II, and we are developing high throughput screens for the assembly pathways (to discover new inhibitors.)

Cytochrome c

Heme protein biogenesis, with emphasis on the cytochrome c biosynthetic pathways.

c-type cytochromes are assembled at their site of function

Genetics and Screens

Rhodobacter capsulatus requires c-type cytochromes for photosynthetic (anaerobic) growth but not aerobic growth. Therefore is a screen for cytochrome c.

The classic "oxidase test" is a secondary screen

TMPD screen for cytochrome c defect in system II: in 1996, respiratory genomics told us that Bordetella would be a good model, leading to isolation of mutants in system II.

Top of the Page

Topology & Functions

CcsB topology by PhoA analysis

Beckett et al. 2000, Mol. Micro

System II topology and mechanism

CcsBA topology (from Helicobacter) and proof of a heme channel; Frawley and Kranz, PNAS, 2009; Kranz et al. MMBR 2009.

System I

CcmF topology and function (Goldman et al. PNAS 1998 and Richard-Fogal et al, EMBO J, 2009 and Kranz et al MMBR 2009)

Biochemistry

Recombinant system I and II - Covalently bound heme is detected with a heme stain after SDS PAGE (Feissner et al. Mol Micro 2006)

Other Biochemistry, including purification of proteins in system I and II can be found in PNAS(2009) and EMBOJ(2009)

Evolution

System I produces more cytochrome c than system II when heme is limited

System I makes cytochromes c even when heme biosynthes is completely inhibited due to the heme reservoir on CcmE. We conclude that system I has advantages when environmental iron (therefore heme) is limiting.

Plant Studies

Reduced trichomes of a T1 CPC responder transformed into a driver line compared to Columbia.

Trichormes can be seen on the leaves of the wild type plant. These are reduced in number on the transgenic line. Luminescence and GUS results are shown for a CPC driver line.

Conclusion: The CPC gene under the control of GAL4 reduces trichome number. Reporter expression correlates to the CPC gene phenotype.

Different transgenic lines were generated with different CPC overexpression patterns

Is the anthocyanin repression physiologically caused by CPC in response to N stress?

Increasing CPC expression levels

Acknowledgements