Download LS1a Fall 09

LS1a Fall 2014
I.

Section Week #9
Membrane transport and membrane potential
Small nonpolar molecules (e.g., O2, benzene) and small uncharged but polar molecules (e.g.,
water) can diffuse through the lipid bilayer without assistance. Large or charged molecules
cannot diffuse freely across the membrane (e.g., amino acids, ions, glucose) and require special
transport proteins.

There are two main types of membrane transport: Passive transport describes the transport of
substrates down a concentration and/or electrochemical gradient, such that no additional energy
is required. Active transport involves transport of substrates against their concentration and/or
electrochemical gradients with the use of energy.

The membrane potential of a cell results from a small net imbalance of positive and negative
ions on the two sides of the membrane.

Potassium leak channels selectively allow potassium ions to diffuse down their concentration
gradient out of the cell, leaving behind a net negative charge inside the cell and providing the
outside of the cell with a slight positive charge. By allowing potassium ions to flow out of the cell,
potassium leak channels are largely responsible for the negative resting membrane potential that
exists in most mammalian cells. The resting membrane potential is reached when the
concentration and electrical gradients that act on an ion are equal and opposite.

The Na+/K+ ATPase pump is responsible for establishing and maintaining the concentration
gradient for Na+ and K+ ions across the plasma membrane. It uses energy from ATP hydrolysis to
actively transport both ions against their concentration gradients. The pump exports three Na+
ions for every two K+ ions it imports.
Section Activity #1: Cells maintain significantly different intracellular and extracellular ion
concentrations.
a. Are the following ions more concentrated on the inside or outside of the cell?
Na+
Higher Inside
Higher Outside
K+
Higher Inside
Higher Outside
Cl
Higher Inside
Higher Outside
b. In most mammalian cells, the intracellular concentration of calcium ions (Ca2+) is tightly
controlled and maintained constant at about 0.0001 mM. The extracellular Ca2+ concentration
in many mammals is close to 2.5 mM. What would be the sign of the membrane potential
(inside with respect to outside) in a cell where only Ca2+-selective ion channels were open and
all other ion channels were closed?
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c. Would the sign or magnitude of this potential change if the extracellular Ca2+ concentration
were 10 mM instead of 2.5 mM? If so, how it would change?
The sign would remain unchanged, but the magnitude would increase. Increasing the
concentration of extracellular calcium results in a larger concentration gradient, which
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c. Would the sign or magnitude of this potential change if the extracellular Ca2+ concentration
were 10 mM instead of 2.5 mM? If so, how it would change?
II. Protein targeting in eukaryotes
 All proteins are translated by ribosomes that are either freely floating in the cytosol or by
ribosomes that are attached to the endoplasmic reticulum (ER).

Proteins destined for the endomembrane system (which consists of the ER, the Golgi apparatus,
lysosomes, endosomes, and peroxisomes) and for secretion outside the cell must first be
translated by a ribosome attached to the ER.

Movement between the ER and subsequent compartments in the endomembrane system (or
“secretory pathway”) occurs via membrane-bound transport vesicles that are loaded with cargo
and subsequently fuse with other compartments.

The topology of membrane orientation is conserved throughout the secretory pathway.

Signal Sequences are amino-acid sequences within a protein that are necessary and sufficient to
target a protein to its correct organelle.

The first signal sequence that has to be recognized for a protein to enter the endomembrane
system is the ER signal sequence, which is recognized by a protein-RNA complex called the Signal
Recognition Particle (“SRP”). Once the SRP binds a ribosome that is translating a protein with an
N-terminal ER signal sequence, the SRP halts translation until the ribosome is delivered to the ER
membrane.

All subsequent signal sequences that determine which organelle a protein will be targeted to are
recognized by proteins called “cargo receptors.”

Some examples of signal sequences are:
Function
Sequence
+
ER signal sequence
H3N-MMSFVSLLLVGILPWATEAEQLTKCEVPQ-…
ER retention sequence
…-(K/H)DEL-COOMitochondrial localization sequence +H3N-MLSLRQSIRFFKPATRTLCSSRYLL-….
Nuclear localization sequence
…-PPKKKRKV-…

Quality control: the ER prevents proteins that are misfolded or not appropriately oligomerized
from leaving. Misfolded proteins may expose amino acid sequences that the ER can recognize
causing the protein to be retained until it properly folds. Similarly, proteins that require multiple
subunits to properly function can be retained in the ER until all of the appropriate peptide
subunits come together.
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
Proteins destined for the nucleus include a “nuclear localization sequence” (an “NLS”) and are
synthesized by ribosomes freely floating in the cytosol. Adaptor proteins that float in the cytosol
bind to an NLS and shuttle NLS-containing proteins through the nuclear pore into the nucleus.
Section Activity #2: Shown below is a simplified diagram of some of the organelles contained
inside a eukaryotic cell. The organelles are not drawn to scale.
Consider a plasma membrane protein that has been fluorescently labeled so that its location in
the cell can be visualized under a microscope.
a. Where does translation of this protein take place? In which of the labeled compartments
would you expect to observe fluorescence in a cell that is actively translating and expressing
this protein?
b. When this membrane protein is present in the endoplasmic reticulum, would its extracellular
domain face the cytoplasm or the ER lumen?
c. Use the terms shown below to describe the path that a plasma membrane protein would take
from translation to reach its destination. Terms may be used multiple times or not at all.
Lysosome
Plasma Membrane
Vesicles
Golgi Apparatus
Nucleus
Cytosol
Endoplasmic Reticulum
Peroxisome
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Section Activity #3: You have developed a cell-free (“in vitro”) translation system to study the
players involved in the translation of secreted proteins. A series of control experiments are shown
below. Microsomes are vesicles derived from ER membranes. Protein X is a known peptide
hormone that is secreted from cells. You add the indicated components to the in vitro translation
system, incubate for 30 minutes, and then analyze protein production using PAGE, with your
results shown below.
Lane 1 provides evidence that the in vitro translation system is working as evidenced by the
presence of Protein X expression when mRNA is added.
a. What are some components that must be included in order to conduct in vitro translation in a
test tube rather than in a cell?
b. Why is the protein in lane 3 so much shorter than in lane 2?
c. Why are the proteins in lanes 4 and 5 the same size? Where is Protein X located in lane 4
(relative to the ER microsome)? Where is Protein X located in lane 5 (relative to the ER
microsome)?
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Section Activity #4: Schematic representations of three proteins are shown below. The amino acid
sequences highlighted as A-E constitute signal sequences. Identify the function of signal
sequences A-E given the results of the mutations described below.
a. Deleting the amino acids in region B causes Protein 1 to be secreted.
b. Deleting the amino acids in region D causes Protein 3 to be cytosolic.
c. Deleting the amino acids in region B and adding the amino acids of region E to the carboxyterminus of Protein 1 causes it be retained in the ER.
d. Deleting the amino acids in region D and adding the amino acids of region C to the aminoterminus of Protein 3 causes it go to the nucleus.
e. Deleting the amino acids in region C causes Protein 2 to be cytosolic.
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