Characterization of Sac7 as a Negative Regulator of the Rho1-mediated

Clathrin Independent Endocytic Pathway in Saccharomyces cerevisiae

By

Matthew R. Peck

A thesis submitted to The Johns Hopkins University in conformity with the requirements

for the degree of Master of Science in Molecular and Cellular Biology

Baltimore, Maryland

April 2015

©2015 Matthew Richard Peck

All Rights Reserved

Abstract

Although it had, until recently, been thought that clathrin-mediated endocytosis was the

sole mechanism for endocytosis in Saccharomyces cerevisiae, a clathrin-independent pathway

that utilizes the small GTPase, Rho1, was recently discovered. Small GTPases are positively

regulated by GTP exchange factors, or GEFs, and negatively regulated by GTPase activing

proteins, or GAPs. Relatively little is known about Sac7, the primary GAP of this pathway. This

study presents the finding of multiple proteins that physically interact with Sac7 via a yeast two-

hybrid screen and preliminary evidence regarding the potential roles these proteins may play in

the clathrin-independent endocytic pathway.

ii

Acknowledgements

At this time, I would like to thank those who helped me to complete this thesis.

I would like to thank everybody in the Wendland Lab. This would certainly not be

possible without the active support of Dr. Beverly Wendland and Dr. Derek Prosser. I would also

like to thank the other members of the Wendland Lab: Kate Bradford, Kristie Wrasman, Kyle

Hoban, Alex Wade, Sami Lux, Marjon Khairy, Thaddeus Woodard, Josh Katz, Asia Poprawski,

and Lydia Nyasea.

I would like to thank members of the Integrated Imaging Center, for whom I am very

much in debt for their help in microscopy portions of this thesis: J. Michael McCaffery, Bethany

Ackerman, and Erin Pryce.

Finally, I would also like to thank my friends and family, without whose constant support

I would not have been able to attend Johns Hopkins University, let alone complete this thesis.

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Table of Contents

Title Page Page i

Abstract Page ii

Acknowledgments Page iii

Table of Contents Page iv

List of Tables Page v

List of Figures Page v

Introduction Pages 1-4

Materials and Methods Pages 5-8

Results Pages 9-16

Discussion Pages 17-20

Literature Cited Pages 21-25

iv

List of Tables

Table 1: Plasmids used in this study. Page 5

Table 2: Strains used in this study. Page 6

Table 3: Genes and corresponding sequences resulting from the Sac7 yeast two-hybrid screen.

Page 13

List of Figures

Figure 1: Confirmation of successful construction of pGBKT7 plasmids expressing Sac7-Gal4

DBD and Lrg1-Gal4 DBD chimeras. Page 10.

Figure 2: Growth suggesting interaction with Sac7 but not with Lrg1. Page 11

Figure 3: Slow grower growth suggesting interaction with Sac7 and often Lrg1. Page 12

Figure 4: Slow grower growth suggesting interaction with Sac7 and often Lrg1. Page 14

Figure 5: Difference between deletion strain and wild type strain were found to be negligible.

Page 16

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Introduction

Endocytosis is an important evolutionarily conserved process necessary for nutrient

uptake, protein turnover at the membrane, and detection of external stimuli (Toret and Drubin,

2006; Prosser et al. 2011). Clathrin-mediated endocytosis (CME) is the best well characterized

mode of endocytosis in Saccharomyces cerevisiae (Boettner et al., 2011). In fact, until recently,

it was believed that CME was the only endocytic pathway in S. cerevisiae, the budding yeast,

despite the existence of clathrin-independent pathways, like caveolae mediated endocytosis,

micropinocytosis, and phagocytosis, in other organisms (Prosser et al., 2011). However, in yeast

cells lacking clathrin, endocytosis occurs, even if at severely reduced rates, suggesting that

Saccharomyces cerevisiae possessed some clathrin-independent method of performing this

indispensable function (Kaksonen et al., 2005; Newpher and Lemmon, 2006). This observation

contributed to the hypothesis that a clathrin-independent pathway exists in S. cerevisiae.

Clathrin-mediated endocytosis is not possible without cargo-specific adaptors at the

membrane which their cargo of interest and requires to the engage clathrin, cargos, and accessory

proteins. Endocytic adaptor proteins bind these distinct elements together to form a functional

unit. There are four main endocytic adaptor proteins required for endocytosis: the

AP180/PICALM homologues, Yap1801 and Yap1802; and the epsins, Ent1 and Ent2.

Simultaneous deletion of 3 endocytic adaptors does not significantly affect endocytosis, but

simultaneous deletion of all four endocytic adaptor proteins is lethal. However, it is not the

entirety of the endocytic adaptor proteins that is necessary (Maldonado-Baez et al., 2008).

Within Ent1 and Ent2 is an Epsin N-Terminal Homology (ENTH) domain, and within

Yap1801 and Yap1802 is an AP180 N-Terminal Homology (ANTH) domain. The ENTH and

ANTH domains are used to bind the plasma membrane, specifically phosphatidylinositol (4, 5)-

1

bisphosphate (Itoh et al., 2001). These domains regulate Cdc42, a protein important in cell

polarity. However, the ENTH and ANTH domains do not include other sites necessary for

clathrin-mediated endocytic function, like the clathrin-binding motif. Therefore, it was predicted

that, by deleting all four endocytic adaptors and adding in an ENTH domain, clathrin-mediated

endocytosis could be eliminated while retaining viability. Although the resulting yeast cells,

hereafter referred to as 4Δ cells, are temperature sensitive, they are viable and have severe

endocytic defects (Maldonado-Baez et al., 2008). These cells were critical in the discovery of a

clathrin-independent endocytic pathway (Prosser et al., 2011).

High-copy suppressor screens find proteins of interest by overexpressing randomized

proteins and then choosing those proteins that suppress a mutant phenotype. Using a high-copy

suppressor screen to test for growth at 37°C in 4Δ yeast cells, a clathrin-independent endocytic

pathway was discovered that relies on the small GTPase, Rho1 (Prosser et al., 2011). The Rho1

endocytic pathway does a relatively small amount of the total endocytosis performed; therefore,

visualization of the pathway only can be done when clathrin-mediated endocytosis is not

functioning. In the study that discovered the Rho1 endocytic pathway, cells with all four

endocytic adaptors deleted but with an ENTH1 domain added were used. Ste3-GFP was used as

the endocytic cargo to visualize this pathway (Prosser et al., 2011). Ste3 is a constitutively

endocytosed receptor expressed in alpha cells used to detect pheromones of the opposite mating

type (Chen and Davis, 2000). This strategy identified Mid2, Rom1, and Rho1 as important

components of this pathway. (Prosser et al. 2011)

However, Rho1, a Ras-like protein whose deletion is fatal for the cell, is involved in a

variety of functions. These functions include establishing cell polarity, regulating protein kinase

C (Qadota, 1996; Drgonova, 1996), regulating Fks1 for 1,3 β-glucan synthesis (Qadota, 1996),

2

and regulating Skn7 for transcription in response to oxidative stress (Helliwell, 1998). When

bound to GTP, Rho1 is active and can perform one or more of its myriad functions but becomes

inactive when the GTP is converted into GDP. In order to control the activity of Rho1, the cell

employs GTPase activating proteins (GAPs) to encourage GTP hydrolysis by Rho1 and guanine

nucleotide exchange factors (GEFs) to encourage the replacement of a GDP with a GTP. Thus,

GEFs promote activation of their cognate GTPases, while GAPs promote their inactivation.

As described above, Rho1 is involved in a large number of cellular processes. Distinct

mechanisms have evolved to use Rho1 in particular circumstances. Both Rho1 GAPs and Rho1

GEFs have been shown to regulate different aspects of Rho1’s function. As an example, the

Rho1 GAPs, Bem2 and Sac7, but not the Rho1 GAP, Bag7, downregulate the Pkc1-Mpk1

pathway (Schmidt et al., 2002). Similarly, Lrg1 is the only Rho1 GAP involved in 1,3- β-glucan

synthesis (Watanabe et al., 2001).

In its function as a Rho1 GAP, Sac7 is involved in Rho1-mediated endocytosis as a

negative regulator of the pathway (Prosser et al., 2011). It was discovered in a screen for proteins

whose mutation suppressed a temperature-sensitive act1 mutant, and was required for growth at

low temperatures (Dunn and Shortle, 1990). It was found later that the cell wall integrity (CWI)

pathway is downregulated by Sac7-mediated inactivation of Rho1 (Bickle et al., 1998; Lockshon

et al., 2012).

There is evidence that Rho1 and Sac7 functions are linked in cell cycle. This evidence

stems from the first study on Sac7, in which yeast with a Sac7 deletion exhibited a sporulation

defect. Sac7 is a negative of Rho1 in the TORC2 signaling pathways, which are important both

for actin organization and for eukaryotic cell growth (Ho et al., 2008; Pracheil et al., 2012). Sac7

is a negative regulator of Slt2, a protein important in cell cycle progression (Arias et al., 2011;

3

Martin et al. 2000). This is in agreement with the finding that SAC7 overexpression results in

slower growth (Akada et al., 1997). In addition, growth defects of mutants in the cell cycle

regulators Cdc34 and Cdc5 are worsened by Sac7 deletion (Varelas et al., 2006). The master

regulator of the meiotic and mitotic cell cycles, Cdk1, phosphorylates Rho1, Bni1, and Sac7

(Ubersax et al., 2003).

Study of the Rho1 endocytic pathway could have applications in humans. A homolog of

Rho1, RhoA, exists in humans, and can even rescue yeast lacking Rho1 (Qadota et al., 1994),

suggesting that RhoA has similar functions to Rho1 (Prosser, 2012). In addition, a RhoA

endocytic pathway exists in mammals (Ellis and Mellor, 2000; Khelfaoui et al., 2009; Kippert et

al., 2007; Wang et al., 2012). RhoA activation increases clathrin-independent endocytosis in

mammalian cells and is an integral participant in Type II phagocytosis (Ellis and Mellor, 2000).

This pathway is critical for oligodendrocyte function (Kippert et al., 2007), making predictable

the finding that inhibition of this pathway recently was proposed as a remedy for mental

retardation in a mouse model (Khelfaoui et al., 2009). Sac7 deletion also reduces the effect of

αS, the toxin responsible for the symptoms of Parkinson’s disease (Wang et al., 2012).

Despite potential applications for understanding Sac7’s role as a negative regulator of

Rho1-mediated endocytosis, Sac7 structure and function are poorly understood (Schmidt et al,

1997). The protein is 655 amino acids long and has only one predicted domain, the Rho1 GAP

domain, which is approximately 230 amino acids in length. Although Sac7 is involved in Rho1

downregulation in a variety of processes, the regulation of Sac7 has not been well characterized.

Physically interacting proteins of Sac7 are candidate regulators of Sac7, and therefore potential

regulators of the Rho1 endocytic pathway through this negative regulator. Therefore, a yeast

two-hybrid screen was performed to find physical interactors of Sac7.

4

Materials and Methods

Plasmid Construction

In this study, 4 plasmids were produced: pGBKT7-Sac7, pGBKT7-Lrg1, pGBKT7-Bag7,

and pACTII-Rho1. These were constructed using PCR to amplify each gene from the genomic

DNA. Enzymatic digestion at key sites in the base plasmids, pGBKT7 and pACTII, homologous

recombination, and growth on synthetic media selecting for successful homologous

recombination were used to construct the product plasmids. Correct construction was verified by

sequencing, enzymatic digestion, and Western blots. All plasmids used in this study are shown

in Table 1.

Plasmid Details Description Source pBW2053 2µ, URA3 pRS416::ENT2 Prosser et al., 2011 pBW0768 CEN, TRP1 pRS414::ENT1 Laboratory plasmid pBW0778 CEN, TRP1 pRS414::ENTH1 Laboratory plasmid pWB2053 2µ, URA3 YEp24::ROM1 Prosser et al., 2011 pBW2525 2µ, TRP1 pGBKT7.SAC7 This study pBW2526 2µ, LEU2 pGBKT7.LRG1 This study pGBKT7 2µ, TRP1 pGBKT7 Laboratory plasmid pACT2 2µ, LEU2 pACT2 Laboratory plasmid

Yeast Strains

For the yeast two-hybrid screen, the PJ69-4A strain was used. This strain is deficient in

its ability to produce uracil, tryptophan, leucine, histidine, and adenine. Activation at this strain’s

GAL4 promoter will induce the production of histidine and adenine. This was accomplished by

Table 1. Plasmids used in this study.

5

having GAL4 promoters integrated immediately upstream of the HIS3 and ADE2 coding

sequences. All strains used in this study are found in Table 2.

Strain Genotype Source

SEY6210 MATα/a, his3-Δ200 trp1-Δ901 leu2-3, 112 ura3-52 lys2-801 suc2-Δ9 BAR1 Laboratory Strain

BWY2858 MATa/α, Ste3-GFP::KANMX6 Prosser et al., 2010

BWY3399 Matα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, pBW0768 Prosser et al., 2010

BWY3400 Matα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, pBW0778 Prosser et al., 2010

BWY6570 MATα, YDL211C-GFP::KANMX6 This Study BWY6572 MATα, Ste3-GFP::KANMX6, ydl211c::NATMX4 This Study BWY6578 MATα, Ste3-GFP::KANMX6, tda7::NATMX4 This Study

BWY6584 MATα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, Ste3-GFP::KANMX6, ydl211c::NATMX4, pWB0768 This Study

BWY6585 MATα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, Ste3-GFP::KANMX6, ydl211c::NATMX4, pWB0778 This Study

BWY6586 MATα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, STE3-GFP::KANMX6, tda7::NATMX4, pWB0768 This Study

BWY6587 MATα, ent1::LEU2, ent2::HIS3, yap1801::HIS3, yap1802::LEU2, STE3-GFP::KANMX6, tda7::NATMX4, pWB0778 This Study

PJ69-4A MATa, trp1-901, leu2-3,112 ura3-52, his3-200, gal4Δ, gal80Δ, lys2::GAL1-HIS3, GAL2-ADE3, met2::GAL7-lacZ James et al., 1996

Yeast Media

The medium used for growing yeast was always either rich medium (yeast peptone

dextrose [YPD]) or, in cases where a particular plasmid needed to be maintained in a strain,

synthetic medium (yeast nitrogen base [YNB]) lacking leucine, tryptophan, leucine, histidine, or

uracil.

Yeast Two-Hybrid Screen

The yeast strain PJ69-4A was transformed with pGBKT7-Sac7 along with plasmids from

the FRYL genomic library. The FRYL library consists of randomized pieces of yeast genomic

Table 2. Strains used in this study. All strains, excluding PJ69-4A, used in this study are isogenic to SEY6210 except at the indicated loci.

6

DNA of mean length 700 base pairs that produce protein chimeras consisting of the activation

domain of Gal4 combined with the protein resulting from the randomized 700 base pairs (Flores

et al. 1999; Fromont-Racine, M et al, 1997). Colonies able to grow on media lacking leucine,

tryptophan, leucine and histidine were selected, and plasmids from these colonies were isolated.

After retransformation into PJ69-4A with pGBKT7-Sac7 to confirm interaction by repeated

growth on selective media, these plasmids were sequenced. Sequences then were put into Base

Local Alignment Search Tools, or BLASTs, that matched these sequences with intervals from

the yeast genome. If a sequence matched a protein through both a nucleotide BLAST and a

protein BLAST, and was expressed as an in-frame fusion with the GAL4 activation domain, the

protein was declared a match.

Deletion and GFP-Tagging

Deletions and GFP-tagging of candidate Sac7 interacting proteins were accomplished

through genomic integration through PCR (Goldstein and McCusker, 1999; Longtine et al.

1998).

Microscopy and Image Analysis

An inverted microscope, the Axiovert 200 (Zeiss), and a 100x, 1.4 NA Plan-Apochromat

objective lens were used to obtain all images in this study. A Sensicam (Cooke) was used to take

pictures while an X-Cite 120 PC fluorescence illumination system was used to excite Ste3-GFP.

All images were taken with cells in YNB medium, and cells were prepared identically.

Testing for Interaction with other Rho1 GAPs

PJ69-4A cells were transformed with a pGBKT7 plasmid expressing the Rho1 GAP

whose interaction was to be tested. Cells containing the desired pGBKT7 plasmid then were

transformed with candidate interactors from the yeast 2 hybrid screen. Growth on selective

7

media indicated interaction. As a positive control, a pACTII-Rho1 plasmid was co-transformed

with both pGBKT7-Lrg1 and pGBKT7-Bag7.

8

Results

Over 1.3 million interactions were tested using the yeast two-hybrid screen with the

negative regulator, Sac7, as the bait. Figure 1 displays the confirmation of the successful

construction of the pGBKT7-Sac7 plasmid via plasmid sequencing and enzymatic digestion. A

Western Blot was used to show correct expression. In the initial screen, growth of 26 colonies

suggested interaction of prey proteins with Sac7. Of these, 12 colonies’ interaction with Sac7

could not be reproduced upon retransformation of the bait and prey plasmids; therefore, these

colonies were not pursued further. The plasmids of 14 colonies reproduced growth on selective

media after retransformation of the plasmids, which indicated interaction with Sac7. Of these 14,

5 colonies produced what will be referred to as a slow-grower phenotype. Figure 2 shows the

consistent growth of the 9 normal growing colonies and Figure 3 shows the growth of the slow-

grower colonies.

9

Figure 1. Confirmation of successful construction of pGBKT7 plasmids expressing Sac7-Gal4 and Lrg1-Gal4 DBD chimeras. (a) Constructed Sac7 and Lrg1 plasmids were digested using EcoR1, yielding a fragment of the correct size of a little more than 1.5 kilobases. (b) A Western Blot confirmed protein was being expressed at the correct size, 94 kiloDaltons. The primary antibody used was anti-Gal4 DNA Binding Domain. (c) Sequencing of the plasmids confirmed that both chimeras were being expressed.

10

Slow-grower colonies consistently show growth on YNB lacking tryptophan, leucine,

histidine, and adenine (YNB-4), which is indicative of protein interaction with Sac7 in a yeast

two-hybrid screen. However, these colonies grow much more slowly than normal growing

colonies. Growth on selective media varies among slow-grower colonies. However, an average

slow-grower colony produces as much growth in 2 weeks as a normal colony would produce in 2

days.

Plasmids from these colonies were isolated and sequenced. The results from this

sequencing are shown in Table 3. Colonies with normal growth produced sequences that

corresponded to proteins in the yeast genome; all slow-grower colonies produced sequences that

did not correspond to known proteins. The proteins implicated by the normal growers in this

screen to be physical interactors of Sac7 are: Ydl211c, a protein of unknown function that

Figure 2. Growth suggesting interaction with Sac7 but with Lrg1. (a) Diagram of streaking pattern used to test for Sac7 and Lrg1 interaction with proteins of interest (POI) (b) Growth after 2 days on conditions selecting for interaction. All four plasmids showing interaction with Ydl211c are included, each with a number corresponding to order of discovery.

11

localizes to the vacuole; Tda7, a cell-cycle regulated protein of unknown function and the

paralog of Ydl211c; Yat2, a carnitine acetyltransferase important in the G1/S transition; Zds2, a

regulatory protein involved in mitosis; Npr3, a component of the SEA, NPR 2/3, and Im1p

complexes; and Flc3, a putative FAD transporter.

In contrast to the strong effect that the Rho1 GAP Sac7 has on the Rho1-endocytic

pathway, the Rho1 GAP, Lrg1, essentially has no effect on this pathway, despite strongly

influencing Rho1 in other functions (Prosser, unpublished results). Therefore, Lrg1 interaction

was used as a negative control to tentatively test for proteins that are specifically involved in the

Rho1 endocytic pathway. Lrg1 was inserted into a pGBKT7 plasmid, and its correct insertion

was confirmed using enzymatic digestion and sequencing. Correct expression was supported by

a Western Blot, as is shown in Figure 1. None of the nine plasmids that yielded sequences that

corresponded to proteins in the yeast genome produced growth indicative of interaction with

Figure 3. Slow grower growth suggesting interaction with Sac7 and often Lrg1. (a) Diagram of streaking pattern used to test for Sac6 and LRg1 interaction with proteins of interest. (b) Growth after two weeks on conditions selecting for interaction.

12

Lrg1. However, of the five slow-grower colonies, four produced growth that indicated

interaction with Lrg1. Thus, the chimeras expressed by the colonies were likely promiscuous in

their physical interaction.

The interacting regions of the Sac7 physically interacting proteins were also identified.

Given the amino acid sequences that resulted from plasmid sequences, it was straightforward to

line these up with the known sequence in the genome. These results are shown in Table 3. NPR3,

had its predicted Sac7 binding region found in a predicted non-cytoplasmic region. Because Sac7

is a cytoplasmic protein, NPR3 was discarded as a potential regulator of the Rho1 endocytic

pathway through Sac7. Ydl211c, which was contained on four candidate plasmids, suggesting

physical interaction Sac7. All four sequences derived from these colonies overlapped with a

region begins with amino acid 330 and ends with the final amino acid at the C-terminal end.

The first four colonies’ plasmid sequences all corresponded to the protein Ydl211c.

Pictures taken of cells expressing GFP-tagged Ydl211c are shown in Figure 4. Previous studies

suggested that Ydl211c localizes to the vacuole (Huh et al., 2003). In my hands, Ydl211c

Table 3. Genes and corresponding sequences resulting from the yeast two hybrid screen.

13

localized to the cell surface, suggesting that it localizes both to the vacuole and to the cell

surface.

Among the proteins found in the screen were Ydl211c and its paralog, TDA7. These two

proteins are related to mucins, which are proteins involved in the production of gels, notably

mucus. Mucins often have a transmembrane domain, a highly glycosylated extracellular domain,

and a cytosolic domain. Both TDA7 and Ydl211c are predicted to have a transmembrane

segment, a cytosolic domain, and an extracellular domain, which one could predict to be

glycosylated based on the proteins’ relation to mucins.

Figure 4: GFP-Tagged Ydl211c suggest localization to the membrane and vacuoles. Saccharomyces cerevisiae cells were subjected to genomic integration via targeting of the endogenous YDL211C gene by which GFP was added to the C-terminal end of Ydl211c and visualized using a fluorescent microscope.

14

Neither TDA7 nor Ydl211c were shown to play a significant role in Rho1 mediated

endocytic activity, as is demonstrated in Figure 5. A strain lacking deletions to Sac7 interacting

proteins shows high endocytic activity for the WT + Vector and 4Δ + Ent1 + Vector cells, poor

endocytic activity for the 4Δ + ENTH1 + vector cells, and improved endocytosis for the 4Δ +

ENTH1 + Rom1 cells. If Tda7 or Ydl211c act as negative regulators of the pathway via Sac7, in

a strain with the protein of interest deleted, the 4Δ + ENTH1 + vector cells would show

improved endocytosis. Similarly, these proteins would be identified as positive regulators should

the 4Δ + ENTH1 + Rom1 cells show poor endocytic activity. However, as Figure 5 shows, this

was not the case.

As was expected, all samples of cells in the far right column, WT +Vector, showed strong

endocytosis, as is demonstrated in the small amount of Ste3-GFP found at the cytoplasmic

membrane. Similarly, the column second from right, showing the 4Δ + Ent1 + vector cells, has

universally strong endocytosis. This was expected because Ent1 restores Clathrin Mediated

Endocytosis. The third column is the first column in which endocytosis was dependent on the

Rho1 endocytic pathway. In all three backgrounds, WT, ydl211cΔ, and tda7Δ, endocytosis was

weak, with much of Ste3-GFP remaining at the membrane. The addition of Rom1 in the fourth

column rescued cells of all three backgrounds to the same comparatively strong endocytic state.

There was no appreciable difference between the three backgrounds, so this experiment failed to

show involvement by Ydl211c or Tda7 in the Rho1 endocytic pathway.

15

Figure 5. Difference between deletion strain and wild type strain was found to be negligible. Ste3-GFP localization was compared in wild type, 4Δ + Ent1, 4Δ + ENTH1, and 4Δ + Rom1 in each of 3 backgrounds: wild type, ydl211cΔ, and tda7Δ.

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Discussion

Although slow-grower colonies initially were regarded as candidate Sac7 interactors, no

useful sequences were derived from them. Nine normal growing colonies and five slow-grower

colonies showed reproducible growth. All nine normal growing colonies yielded sequences that

corresponded to genes in the yeast genome and none of the five slow-grower colonies produced

such sequences. In addition, 4 of the 5 slow-grower colonies showed growth indicative of

interaction with Lrg1, suggesting that the interaction lacked protein/pathway specificity.

Therefore, we suggest that colonies of this slow-grower phenotype be disregarded in future yeast

two-hybrid screens.

TDA7 and YDL211C are paralogs, and their expression is regulated by the cell cycle.

Tda7 is known to physically interact with Rvs167, Lsb3, and Sla1, which are all involved in

clathrin-mediated endocytosis (Tonikian et al., 2009). Clathrin-mediated endocytosis consists of

three phases: the early immobile phase, the mid/late immobile phase, and the mobile phase

(Boettner et al., 2012). During the immobile phase, Ede1 and clathrin are recruited to the site of

endocytosis (Toret and Drubin, 2006). During the mid/late immobile phase, Las17p is recruited

to the site about 2 minutes after clathrin arrives, and recruits the Arp2/3 complex, which is

required for actin assembly (Boettner et al., 2012; Toret and Drubin, 2006). Notably, Lsb3, one

of the proteins that binds to Tda7, binds Las17. At the same time that Las17 is being recruited to

these sites, the Pan1 complex, which includes Sla1, a second protein known to bind with Tda7,

also is being recruited. Next, Vrp1 and Myo5 are recruited to the site and the process of actin

assembly begins. Internalization of the coat then begins, thus starting the mobile phase. The

forming vesicle must be excised from the membrane, and this is accomplished by Rvs161 and

Rvs167 (Boettner et al., 2012; Toret and Drubin, 2006), a third protein found to bind with Tda7.

17

Thus, Tda7 is known to bind with proteins from the early stages of the clathrin-mediated

endocytic pathway to the end stages. Because Sac7 is a negative regulator of the Rho1 endocytic

pathway, it is tentatively hypothesized that Tda7 plays a role in segregating of the two endocytic

processes. It is possible that Ydl211c, its paralog, could have a similar function.

Npr3 also was found to physically interact with Sac7. Npr3 acts as a part of three

different complexes: the Npr2/3 complex (Neklesa and Davis, 2009), the SEA complex

(Dokudovskaya et al., 2011), and the Iml1p complex (Wu and Tu, 2011). Interaction with Sac7

could, therefore, be relevant for any of these three complexes. The Npr2/3 complex mediates

cellular response to amino acid starvation through the TORC1 pathway (Neklesa and Davis,

2009). Amino acid starvation certainly could have effects on cellular decision making regarding

endocytic function and cell wall maintenance, both of which Sac7 negatively regulates. This

interaction could result in reduced Rho1-mediated endocytosis and less emphasis on cell wall

maintenance in response to amino acid starvation. The SEA complex associates with the vacuole

and is involved in intracellular trafficking (Dokudovskaya et al., 2011). It is possible that an

endosome produced by Rho1 endocytosis could use the SEA complex to attract Sac7 to itself and

thereby downregulate any remnant endocytic machinery on the endosome. The Iml1p complex

acts to regulate non-starvation induced autophagy in the cell (Wu and Tu, 2011). Sac7’s role in

the CWI pathway could be involved here. When the Iml1p complex is acting to induce

autophagy in the cell, actions of the CWI pathway upon the object of the autophagy would be

non-constructive. Therefore, it is conceivable that the Sac7 could act with the Iml1p complex to

downregulate the CWI pathway when autophagy is being performed.

Sac7’s interaction with Yat2 also could have biological significance. Yat2 is a carnitine

acetyltransferase that is found in the mitochondria (Swiegers et al., 2001). Carnitine

18

acetyltransferases are important for the transition from the S phase to the G1 phase in the cell

cycle (Brunner et al., 1997). During the cell cycle, the cell wall undergoes substantial

reconfiguration in order to produce the final product of two independent cells. Therefore, there

must be mechanisms to coordinate the CWI pathway with the progress of the cycle. Yat2’s

physical interaction with Sac7 could be one method of doing this. Lastly, a Yat2-Sac7 interaction

could stop the CWI or Rho1 endocytic pathways from occurring at the mitochondria, or it could

be unrelated to endocytosis.

Flc3 interacting physically with Sac7 potentially is meaningful as well. Flc3 is found in

the endoplasmic reticulum (Sickmann et al., 2003; Protchenko et al., 2006), where it acts as a

flavin adenine dinucleotide transporter (Protchenko et al., 2006) similar to its paralog Flc1

(Byrne and Wolfe, 2005). The Flc3-Sac7 physical interaction could simply help ensure that the

CWI and Rho1 endocytic pathways are not active at the ER. However, deletion of FLC genes

results in poor assembly of the cell wall, thus displaying a role of FLC3 in the cell wall

(Protchenko et al., 2006). Given that Flc3 acts to support cell wall assembly and Sac7 acts to

suppress the CWI pathway, the physical interaction between the two ensures that the processes

of cell wall assembly and the CWI pathway are segregated from each other and therefore do not

interfere with each other.

Of the 6 proteins derived from this screen, two of them, Tda7 and Ydl211c, are paralogs.

Therefore, paralogs of other proteins found in the Sac7 yeast two-hybrid screen may be strong

candidates for physical interaction with Sac7. Of the remaining 4 proteins, two, Flc3 and Zds2,

have paralogs, Flc1 and Zds1, respectively. Future tests for physical interaction with Sac7 would

be remiss to not include testing for the potential interaction between Flc1 and Zds1 with Sac7.

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Bag7 is also a Rho1 GAP. It had been shown previously that Bag7 and Sac7 act together

in some functions, like cold-sensitivity suppression, but not in others. (Schmidt et al., 2002)

Additionally, Sac7 and Bag7 are paralogs, and could share other functions. Therefore, we predict

that some of the Sac7 interacting proteins identified in this study would be found to interact with

Bag7, so future studies of these proteins would be wise to include study on interaction with

Bag7.

Although 5 proteins were found in this study to interact with Sac7, only two, Ydl11c and

Tda7, were tested for involvement in the Rho1 endocytic pathway. Due to time constraints, the

potential for Flc3, Yat2, or Zds2 involvement in the pathway was not tested. Future studies

should investigate these three proteins as potential regulators of the pathway through Sac7.

The functions of the various domains of Sac7 are not well characterized. Although this

current study successfully found 6 proteins that physically interact with Sac7, characterization of

Sac7 did not determine which domains of Sac7 were binding these proteins. Therefore, future

work could concern which parts of Sac7 were involved in binding the proteins found in the

study. Because the only known domain of Sac7 is the Rho1 GAP domain (Schmidt et al, 2002),

preliminary study likely would break Sac7 into the pre-Rho1 GAP domain, the Rho1 GAP

domain, and the post Rho1 GAP domain. Future studies will continue to elucidate more about

the regulation of the Rho1 endocytic pathway, allowing for clearer understanding of endocytosis

in yeast, endocytic processes in general, and perhaps allowing for applications in higher order

animals, including humans.

20

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Curriculum Vitae

Matthew Richard Peck

3027 Decatur Street, West Lafayette, Indiana 47906

mpeck6@jhu.edu; (765) 714-9806

EDUCATION

• Honors Core 40 Diploma with Academic Honors, West Lafayette High School, West Lafayette, Indiana, 2011. GPA: 4.0

• Bachelor of Science, Arts and Sciences, Biology, The Johns Hopkins University, Baltimore, Maryland, 2014 GPA: 3.7

• Master of Science, Arts and Sciences, Biology, The Johns Hopkins University, Baltimore, Maryland, 2015

AWARDS

• Dean’s List: Fall 2011, Spring 2012, Fall 2012, Spring 2013, Fall 2014 • Beta Beta Beta Honor Society, Associate Member and President: April 2013

RESEARCH EXPERIENCE

• Research Assistant, Wendland Lab, Department of Biology, The Johns Hopkins University, Baltimore, Maryland. August 2012 to present.

POSTER PRESENTATIONS • Peck Matthew, Prosser Derek, Wendland Beverly. Preliminary Characterization

of Sac7 as a Negative Regulator of the Rho1-mediated Clathrin Independent Endocytic Pathway in Saccharomyces cerevisiae. Poster presented at Beta Beta Beta poster session, The Johns Hopkins University, April 2014, Baltimore, Maryland.

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