Biochemical analyses of the Golgi and its associated membrane proteins in Saccharomyces cerevisiae

Secretory trafficking in eukaryotic cells occurs via membrane-enclosed organelles and vesicular intermediates which deliver proteins and lipids to their cellular destinations. Throughout this process, the unique nature of organelles is likely to be maintained through: (i) membrane recycling events that function in the retrieval of protein and lipid components from later stages in the pathway, and (ii) compartment-specific scaffolding factors that act in securing the structural and spatial integrity of the organelle. The Golgi apparatus of the yeast Saccharomyces cerevisiae has been defined through the use of conditional mutants which accumulate aberrant Golgi structures under restrictive conditions. This manuscript defines a novel enrichment scheme for the purification of these aberrant structures and identifies two proteins as steady-state residents of the yeast Golgi. These resident proteins have been used for the enrichment of Golgi from wild-type cells. Further, a series of monoclonal antibodies were generated to carbonate insensitive proteins from this membrane population in an attempt to identify additional compartment-specific factors. Four of these monoclonal antibodies recognized protein species of 51 kDa, 36 kDa, 95 kDa and 77 kDa. Each of these proteins displayed carbonate insensitivity and only the 95 kDa species was found to be glycosylated. The Golgi-specific residence of each of these proteins was established by indirect immunofluorescence and quantitative biochemical fractionation. In addition, analyses of conditional secretory (sec) mutants blocked at various stages in the secretory process suggested that the 51 kDa species progressed to the Golgi but was not incorporated into post-Golgi secretory vesicles. These data were consistent with the proposed intracellular localization of these proteins to the yeast Golgi.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Whitters, Eric Anthony

• Biology, Molecular
• Biology, Cell
• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1994 Whitters, Eric Anthony

Language dc:language

eng

Identifier

AAI9503351
(UMI)AAI9503351

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/20283