Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Author ORCID Identifier

https://orcid.org/0000-0003-4238-9088

Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

Year Degree Awarded

2019

Month Degree Awarded

September

First Advisor

Magdalena Bezanilla

Subject Categories

Cell Biology | Genetics | Molecular Biology | Plant Biology

Abstract

Polarized secretion is essential for cell biological processes across the tree of life. In particular walled organisms, such as yeast, filamentous fungi, and plants use polarized secretion to divide and grow thereby providing excellent model systems to unravel the molecular mechanisms controlling this process. For polarized growth, an active secretory system is required to deliver specific building materials to the growth region. As part of the secretory pathway, transport from the endoplasmic reticulum (ER) to the Golgi apparatus, which is mediated by the coat protein complex II (COPII) is therefore a prerequisite for polarized secretion. I have investigated the role of COPII in the juvenile tissue of the moss Physcomitrella patens, which grows exclusively by polarized secretion. Interestingly in P. patens, small gene families encode each of the subunits of the COPII complex. To investigate whether these small gene families are redundant, I analyzed the loss of function phenotype resulting from silencing Sec23, a component of the inner coat of COPII, which is encoded by seven highly similar genes. To my surprise, I found that silencing Sec23D caused a severe growth defect, while silencing the remaining six Sec23 genes had no significant defect. Coding sequence complementation showed partial functional redundancy between only Sec23D and Sec23E, suggesting that Sec23D and to a limited extent Sec23E play pivotal roles during polarized secretion, I found that Sec23D localizes to the ER and partially overlaps with the Golgi. In addition, Sec23D heterodimerizes with two of the seven Sec24 genes in moss, suggesting that Sec23D is a canonical COPII component. To analyze the role of Sec23D and the remaining six Sec23 isoforms, I developed CRISPR-Cas9 mediated mutagenesis vector system for P. patens. This genomic editing method was shown to be more efficient than traditional homologous recombination. Using stable sec23 mutants, I found that the sec23d null mutants grow very slowly, have significant defects in ER morphology, and in secretion to the Golgi as well as to the plasma membrane. However, I observed that ∆sec23abcfg mutants grow similar to wild type, have no ER morphology defects, no secretion defects to the Golgi, and only a mild secretion defects to the plasma membrane. These data suggest that Sec23D is the dominant Sec23 subunit of the COPII complex, mediating the majority cargo transport and the specific cargo transport from the ER to the Golgi during polarized growth. Using CRISPR-Cas9 genome editing, I uncovered an interesting in-frame deletion mutant, sec23d∆AAL, which resembles the sec23d null mutant. Like the sec23d null mutant, sec23d∆AAL exhibits severe growth defects, altered ER morphology, and defects in secretion to the Golgi as well as to the plasma membrane. Imaging mRuby2 tagged sec23d∆AAL revealed that sec23d∆AAL is expressed but may not function like the wild type protein. To investigate how loss of these three residues impacts the function of Sec23D, I performed a directed yeast two-hybrid assay to demonstrate that the deletion mutant no longer binds Sec24C. To determine if this site is conserved in other organisms, I deleted the equivalent amino acids in budding yeast and human Sec23 proteins (ScSec23 and HsSec23A). In a yeast complementation assay, where the wild type copies of ScSec23 and HsSec23A rescue the yeast sec23-1 temperature sensitive mutant, I found that mutants in ScSec23 and HsSec23A harboring either deletion of the equivalent three amino acids or conversion of those amino acids to glycine were unable to rescue. These data suggest that these residues in the N-terminus of Sec23 are evolutionally conserved and allosterically regulate the interaction between Sec23 and Sec24.

DOI

https://doi.org/10.7275/trjm-xr33

Share

COinS